Psg) genes encode proteins of unknown function, and are members of the carcinoembryonic antigen (Cea) gene family, which is a member of the immunoglobulin gene (Ig) superfamily. In rodents and primates, but not in artiodactyls , there have been independent expansions of the Psg gene family, with all members expressed exclusively in placental trophoblast cells. For the mouse Psg genes, we sought to determine the genomic organisation of the locus, the expression profiles of the various family members, and the evolution of exon structure, to attempt to reconstruct the evolutionary history of this locus, and to determine whether expansion of the gene family has been driven by selection for increased gene dosage, or diversification of function.The pregnancy-specific glycoprotein (Psg gene sequences currently in the public genome and expressed-sequence tag (EST) databases and used systematic BLAST searches to generate complete sequences for all known mouse Psg genes. We identified a novel family member, Psg31, which is similar to Psg30 but, uniquely amongst mouse Psg genes, has a duplicated N1 domain. We also identified a novel splice variant of Psg16 (bCEA). We show that Psg24 and Psg30 / Psg31 have independently undergone expansion of N-domain number. By mapping BAC, YAC and cosmid clones we described two clusters of Psg genes, which we linked and oriented using fluorescent in situ hybridisation (FISH). Comparison of our Psg locus map with the public mouse genome database indicates good agreement in overall structure and further elucidates gene order. Expression levels of Psg genes in placentas of different developmental stages revealed dramatic differences in the developmental expression profile of individual family members.We collated the mouse Psg exon organization, the mouse Psg genomic locus structure, and the expression patterns of individual Psg genes. This information will facilitate functional studies of this complex gene family.We have combined existing information, and provide new information concerning the evolution of mouse  In mammalian pregnancy the interaction between the maternal uterine tissues and foetal trophoblasts is regulated by a wide variety of cellular and endocrinological mechanisms. These mechanisms underpin trophoblastic invasion and remodelling of maternal tissues, placental angiogenesis, and the modulation of maternal immune responses. Central to these processes is the production by trophoblast of a variety of hormones that are found in abundance in the maternal bloodstream during pregnancy .The pregnancy-specific glycoproteins (PSG) are the most abundant foetal proteins in the maternal bloodstream in late pregnancy . They arThere are 11 members of the PSG family in humans that are encoded by genes clustered on chromosome 19q13.2 . PSG proH1 response to a predominately TH2 response which is more compatible with a successful pregnancy [The biochemical properties and physiological functions of the PSGs remain to be fully elucidated, although functional experiments and clinical observations are beginning to provide some clues. Low PSG levels in the maternal circulation are associated with threatened abortions, intrauterine growth retardation and foetal hypoxia -19. The regnancy .The only PSG receptor identified to date is the integrin-associated CD9 receptor, which was found to bind the N1 domain of both PSG17  and PSG1Psg locus, which we undertook to produce in the work described herein. It is also pertinent to ask whether the independent expansions of PSG gene families in different mammalian lineages reflect selection for increased gene dosage or for diversification of function mediated through different protein structures or developmental expression patterns. We therefore undertook to examine and correlate protein domain evolution and expression profiles of the various mouse Psg genes to attempt to address this question. Our results suggest that different family members have very different expression levels at different stages of development, which we consider may be supportive of the hypothesis that mouse Psg genes may have evolved divergent functions in mammalian pregnancy. However, mutagenesis of individual family members will be necessary to rigorously test this hypothesis.Much current work has focussed on human PSGs due to their possible relevance to disorders of pregnancy. However, the study of rodent PSGs is important because, the evident differences between primate and rodent PSG protein domain structures notwithstanding, there appears to be considerable conservation in terms of expression in trophoblast, independent gene family expansions in mammalian lineages with haemochorial placentation, and postulated immune functions during pregnancy. Moreover, the application of gene targeting and mutagenesis in the mouse is likely to be informative with respect to elucidating the cellular and physiological functions of PSGs. Such experiments will require an accurate genomic map of the mouse Psg17, Psg18, Psg19, Psg21, Psg23, Psg28 and Psg30 were identified via basic name searches of the RefSeq RNA database. Their identity was then verified by comparison to cDNA fragment sequences, which were obtained during the course of this work and deposited in GenBank [Psg22 was then identified via BLAST analysis of the mouse RefSeq RNA database using the GenBank partial sequence referenced in Beauchemin et al. [Psg31 was identified by BLAST analysis of the same database using the full-length Psg30 sequence and found to be the XM_355864.1 predicted transcript. However, there was a discrepancy between the predicted transcript and the sequences of EST clones CK032208 and CN694284. Comparison of these EST sequences with genomic contig NT_039395.2, using pairwise BLAST analysis, revealed that there had been a duplication of the N1 domain exon. We refer to the two N1 domains of Psg31 as N1 and N1* hereafter.For comparative studies of the human PSG family it is relatively easy to compare coding sequences (CDS) and peptide sequences because complete sequence information is available. However the data available for mouse PSGs is not complete, making such analyses difficult. Thus, we firstly collated the currently available public data, and we then attempted to identify sequences for PSGs that were not completely resolved in the databases. Full-length cDNA sequences of  GenBank , as misnn et al. . Psg31 wPsg20, Psg24, Psg25, Psg26, Psg27 and Psg29) were deduced manually by systematic BLAST analysis of the mouse genome database as described in Methods. None of these predicted cDNAs were observed in the mouse EST database, although all except Psg20 were observed in the Trace Archive EST sequences. A novel splice variant of Psg16 was also found. BLAST analysis of the mouse High Throughput Genomic Sequences (HTGS) database identified contig AC148976.2, which appears to contain the whole Psg16 gene. An alternative exon 1 was discovered upstream of the previously described initiating exon by a pairwise BLAST comparison of this contig with full-length Psg17 coding sequence. The use of this alternative exon 1 produced a transcript that encodes a typical PSG polypeptide complete with a predicted secretory-peptide signal sequence and cleavage site. Multiple hits identified from subsequent BLAST analysis of the mouse EST and Trace Archives EST databases provided evidence that this novel splice variant was placentally expressed in vivo. In contrast, only one hit was obtained by identical analysis using the coding sequence of the brain-specific transcript described in Chen et al. [Psg16. Unlike the brain-specific variant, the placentally-expressed variant possesses a predicted secretory signal peptide at the N-terminus, like most other Psg gene family members.The gene and full-length cDNA coding sequences of the remaining mouse genes  also revealed differences in the encoding of the A-domain. The placental transcript is predicted to be encoded by 5 exons, as are the majority of mouse Psg mRNAs. However, a weak splice donor signal sequence within the fifth exon permits splicing to a strong splice acceptor sequence downstream of the sixth exon, as seen in the brain-expressed transcript. Trace Archive EST data reveals multiple hits to sequences from placental cDNA libraries using the 3' end of the placental Psg16 coding sequence as bait. This confirms the existence of our predicted transcript. Conversely, similar analysis using the brain-expressed variant yielded no hits. The sixth exon is present on a separate randomly ordered gene fragment within the AC148976.2 contig.The comparison of the brain derived Psg-ps1 was previously considered to be a pseudogene, based on a point deletion at nucleotide position 30, downstream from the canonical Psg translational start site [Psg continues 105 bp upstream of the site of the mutation to an alternative ATG. Inspection of the sequence revealed a Kozak consensus, and BLAST analysis of the public EST and Trace Archive EST databases yielded many mRNA clones that contain this region in addition to downstream exons. Hence, this gene is clearly expressed, and we now propose to rename Psg-ps1 as Psg32 hereafter. We note that this mutation and amino terminal extension abolishes the canonical PSG secretory signal and peptide cleavage site. We therefore suggest that if Psg32 is indeed translated, the resulting protein is retained within the cytoplasm. To determine if the deletion observed in BALB/c mice was also present in other murine strains, we amplified and sequenced a 146 bp fragment by PCR using a set of primers specific for the 5'-untranslated region and the leader peptide of Psg32. The deletion observed in the Psg32 cDNA is also present in the genomic DNA of A/J, C57BL6/J, YBR/Ei, and SWR/J inbred mouse strains (data not shown).art site . HoweverPsg16, Psg20, Psg26 and Psg31 are listed in Psgs (except the brain-specific splice variant of Psg16) are listed in Psg16) are listed in The nomenclature (past and current) and accession numbers of nucleotide sequences of all the murine PSGs are documented in Table Psgs, thirteen encode a common structure of three Ig variable (IgV)-like domains (N-domains) and a single Ig constant (IgC)-like domain (A-domain). Psg24, Psg30 and Psg31 have an expanded structure created by the duplication of IgV-like domains. An unrooted phylogenetic tree indicates three main branches of IgV-like domain evolution  trees with bootstrap values of 1000 were prepared  within which the order of Psg genes was determined to the fullest extent possible. The orientation of the two subclusters with respect to each other and the chromosome 7 centromere was determined by fluorescent in situ hybridisation (FISH) analysis. These data are summarised in Fig. Psg genes are located within cytobands A1 and A2 on proximal chromosome 7 and are interspersed with other genes, particularly Ceacams, as determined by comparison with the published mouse genomic sequence on contig NT_039395. We did not observe any obvious correlation between the relative positions of the Psg genes at the locus and their domain arrangements or expression patterns.The published mouse Psg29 and Psg32. However, we determined that the subclusters are fused between Psg32 and Psg30/Psg18. YAC F10104 (which is non-chimeric) is about 460 kb long and contains only two Psg genes which indicates the presence of a non-Psg genomic region. We estimate the gap between the subclusters to be approximately 400 kb based on the size of cosmids containing two Psg genes (ca. 40 kb).There is a discrepancy with respect to the distance between the two subclusters. The currently poorly resolved data covering this region in the the Ensembl assembly implies the presence of a gap between PSG genomic data in the public databases are relatively complete. For each PSG gene, there are annotated RefSeq resources comprising information on genomic structure, transcripts and translation products. The nomenclature is also standardised [PSG locus and surrounding genes. In contrast, a substantial quantity of mouse Psg genomic data in the public domain is fragmented, incomplete and somewhat unreliable. We sought to collate the existing genomic data, to present novel data to fill in gaps, and to provide a coherent resource of mouse Psg genomic data.The human dardised . FurtherPsg genes was complete we performed systematic BLAST searches of a variety of public DNA sequence databases. This analysis revealed the existence of a novel expressed Psg gene, which we name Psg31 in line with the accepted nomenclature convention [Psg31 apparently evolved from a duplication of the whole of the Psg30 gene followed by a subsequent internal duplication of the N1 domain. We were also able to predict the complete coding sequences of four Psg genes for which previously only partial fragments were described. The gene, CDS and protein sequences of these predictions, coupled with a complete reference of all known mouse Psg CDS and primary protein sequences are provided in three attached Additional Files.To determine whether the existing set of mouse nvention . Psg31 aPsg gene sequences, domain structures for all family members were predicted. All of the PSG proteins possess previously described arrangements of Ig-like domains. Except for two members, discussed below, all are predicted to encode N-terminal secretory signal sequences. Our predicted novel splice variant of Psg16 has a complete N1 domain and secretory signal peptide sequence. Trace Archive EST database BLAST analysis confirmed that this variant is expressed in the placenta. In contrast, the brain-expressed variant [Psg-ps1 pseudogene [Psg32, is not a pseudogene but a bona fide expressed Psg gene family member. The Psg32 transcript may encode a protein that is retained within the cytoplasm. We note that a precedent in the human exists in the form of a non-secreted splice variant of PSG11 [Using the full CDS information obtained for the complete set of mouse  variant  has onlyeudogene  was founof PSG11 .Psg31 has the unusual N1-N1*-N2-N3-N4-N5-N6-N7-A domain structure. This newly characterised Psg gene has evolved from a duplication of the entire Psg30 gene followed by an internal duplication of the N1 domain. There may be functional significance associated with the N1 domain duplication. The complex nature of Psg gene evolution, including putative gene conversion and recombination events between family members [Psg24, Psg30 and Psg31. We note that the apparent route to domain number expansion differed between Psg24, and Psg30 and Psg31. The extra N-domains of Psg24 are derived from two duplications of the N2 domain. However, for Psg30 and Psg31, independent duplications of each of the N2 and N3 domains were probably followed by a secondary duplication of the daughter domains, possibly as a single event. members , makes iPsg genes. In this analysis Psg21 and Psg23 were the most abundant, consistent with a previous report of abundant Psg23 expression [Psg gene expression differences reflect selection for divergent functions, or increased gene dosage for enhancement of an existing function, because expression levels were uniformly low for many family members, and there was a general trend of increased expression during gestation. Psg transcripts are found from day 6.5 of embryonic development onward in primary trophoblast giant cells, later (from day 10.5) in spongiotrophoblast cells and, to a lesser extent, in a cell population in the deciduas basalis at day 14.5 [Psg genes exhibit different cellular expression patterns, which might indicate divergent functions of the various PSGs. There are interesting parallels between the expansion of the Psg gene family and similar expansions of other placentally-expressed gene families such as the prolactin and growth hormone families [Gene expression data from RT-PCR of placental RNA and EST database analysis revealed considerable differences in the expression levels of different pression . Whilst day 14.5 . At presfamilies . Such dufamilies .Psg gene protein coding sequences and protein domain structures, the mouse Psg genomic locus on chromosome 7 remained to be determined to complete a comprehensive resource for the analysis of Psg function. The NCBI build 32 composite mouse assembly data revealed that only four Psgs had been mapped. Other Psgs on contig NT_039395 are currently unordered. We therefore screened cosmid, YAC and BAC libraries, and orientated Psg-containing clones to identify, where possible, the order of Psg genes within the locus. We were not able to resolve all ambiguities in gene order on our map; however, where public database information is available, our data are in good agreement. We found no clear relationship between gene location and gene expression level suggesting that, within the Psg locus, each Psg gene is autonomously regulated.Having collated all known mouse Psg gene family are poorly understood. This is a feature shared with other placentally-expressed, multigene families such as the prolactin and growth hormone genes [Psg genes, including the generation of mouse mutants with modified Psg gene expression profiles, we have collated the entire set of mouse Psg genes, their predicted encoded proteins, and their evolutionary histories. The complete CDS data will enable the cloning, over-expression, and gene targeting of individual or multiple mouse Psg genes. This will facilitate the elucidation of their function and, by extrapolation, their human homologues, which may be involved in diseases of pregnancy.The evolution and physiological functions of the relatively understudied mouse ne genes . In ordePsg gene-containing cosmids using a 32P-labelled, full-length Psg cDNA , 0.1% sodium dodecyl sulfate (SDS) at 65°C. The membranes with arrayed YAC clone DNAs were kindly provided by Dr. H. Lehrach, Max-Planck-Institut für Molekulare Genetik, Berlin. Two libraries were screened, 902 and 903, both in the vector pYAC4 [32P-labelled probe consisting of the N1 domain exon sequences of 14 mouse Psg genes (Psg17–Psg29 and Psg32). The N1 domain exons were amplified individually by PCR using the degenerate primer pair PsgN1-F/PsgN1-R or Psg32N1-F/PsgN1-R (4 mM each) for Psg32 . N1 exons of Psg28 and Psg29 were released by digestion with SalI and KpnI from pUC18 (see below).Cosmid libraries in pWE15 which were made from liver DNA of BALB/c and C57BL/6 mice were obtained from Dr. Edwin N. Geissler, Boston, MA, USA, and Stratagene , respectively. They were screened for the presence of f pCea2b ) as a prembranes  with theor pYAC4  composedArthrobacter luteus . Two mg of cosmid or 0.5 mg of YAC DNA were digested with restriction endonucleases, size fractionated by electrophoresis on 1% agarose gels and blotted onto positively charged nylon membranes. To identify N1 and A domain exon-containing DNA fragments, the digested DNAs on the membranes were hybridised with 32P-labelled N1 (cosmid DNA blots only) and A domain probes from Psg17 and washed under medium stringency conditions . The Psg17 N1 and A domain cDNA fragments used as probes were obtained by PCR  using the mPsg-5'/mPsg-3'  and Psg17A5'/Psg17A3' , primer pairs respectively, and the Psg17 cDNA clone pCea2b as template  in a total volume of 100 μl using 200 ng of YAC clone DNA as template, 1 U Taq polymerase, 3 mM MgCl2 and 4 mM each of PsgN1-F and PsgN1-R degenerate oligonucleotides (Table Psg genes (except Psg32). The product was purified by electrophoresis on a 1.8% agarose gel and subcloned into pUC18 after blunt-ending . The N1 exons from two of the 10 newly identified Psg genes  were analysed by sequencing recombinant plasmids which did not hybridise with oligonucleotide probes specific for known Psg genes  and cosmid clones (10 ng) were used to amplify the N1 domain exons of all known Psg genes in a total volume of 60 μl as described above. The N1 exon of Psg32 was amplified in a separate reaction using Psg32N1-F and PsgN1-R , gene-specific oligonucleotides  essentially as described previously [The presence of the different m Primer . The onleviously .Psg gene subclusters was defined by FISH analysis using probes described in Fig. The chromosomal location and chimerism of YAC clones were determined by FISH analyses, using B1-PCR of YAC DNA for probe preparation essentially as described . OrientaPsg genes was studied by RT-PCR followed by hybridisation of the products with gene-specific oligonucleotides. Total RNA (1 μg) from placentae of different gestational stages was reverse transcribed in a total volume of 10 μl by avian myoblastosis virus (AMV) reverse transcriptase  in the presence of 6 U/μl RNasin (Promega) using a degenerate oligo (dT)30 oligonucleotide (1 μM) as primer 2SO4, pH 8.6), 3 mM MgCl2 and 0.4 mM dNTPs in a total volume of 100 μl. Amplification of all known Psg cDNAs (except for the cDNA of Psg32 (Cea6), which at the time of the experiment was presumed to be a pseudogene [Taq polymerase after addition of 400 pmoles of Psg-all  or a Taq Dye Deoxyterminator cycle sequencing kit .. Where fully annotated, Psg cDNA sequences were identified by name searches of the RefSeq RNA database. Attempts were then made to identify remaining known Psg cDNA sequences via BLAST analyses of the mouse RefSeq RNA database using the GenBank partial-sequences referenced in [Psg17 cDNA sequence, or fragments thereof, as a probe. A similar procedure was applied to situations where the existence of alternatively spliced exons was suspected to reside within in a Psg gene-containing contig. In the cases where Psg mRNA sequences were built from genomic sequence, or splice variants were predicted, evidence for the existence of such mRNA species in vivo was tested by BLAST analysis using the mouse EST and Trace Archive EST databases.All bioinformatics searches described below used the online software and databases available at the NCBI enced in . Any PSG.The coding sequences of the PSG domains were aligned using Clustal X . For thePsg32-specific oligonucleotide, Psg32-exon 1, which binds to a sequence internal to the PCR primers were sequenced.Four hundred nanograms of DNA obtained from four inbred mouse strains   were amplified using the oligonucleotide primers 5'-AAGGAAGGACAGCAAAT and 5'- AGCTGTGAGCAGAAGAC  with Pfu DNA polymerase (Stratagene) following the manufacturer's instructions. The 146 bp PCR products were subcloned into PCR-Script (Stratagene). Clones that hybridized to a Psg locus organisation, phylogenetic analyses, Psg expression studies, and drafted the manuscript. BF characterized Psg genomic clones and performed Psg expression studies. GD isolated the Psg BAC clones and analysed Psg32 exon 1 sequences in mouse strains. MB and FW contributed to Psg expression studies and YAC fragment assembly. TH and KO performed FISH analysis for identification of orientation of Psg subclusters. TM co-conceived the project and coordinated work performed in Cork. WZ co-conceived the project, coordinated all work performed in Germany, and performed bioinformatics relating to Psg phylogeny and expression.ASM performed bioinformatics relating to Psg16 , Psg20, Psg24, Psg25, Psg26, Psg27, Psg29 and Psg31 The gene sequence and predicted CDS and primary protein sequences for A basic text file containing the primary genome data (with source HTGS or WGS information). Predicted CDS sequence is included along with translations. Reverse complement is abbreviated 'RC' in the text.Click here for filePsg CDS sequences Complete set of mouse A basic text file in FASTA format containing CDS sequences for all known mouse Psgs (not including the brain-specific variant of Psg16).Click here for fileComplete set of mouse PSG primary protein sequences A basic text file in FASTA format containing primary protein sequences for all known mouse Psgs (not including the brain-specific variant of Psg16).Click here for file


DNA techniques, analytical methods and palaeoclimatic studies are greatly advancing our knowledge of the global distribution of genetic diversity, and how it evolved. Such phylogeographic studies are reviewed from Arctic, Temperate and Tropical regions, seeking commonalities of cause in the resulting genetic patterns. The genetic diversity is differently patterned within and among regions and biomes, and is related to their histories of climatic changes. This has major implications for conservation science. Phylogeography, named by Avise et al in 1987 ,3. GenetWhilst mtDNA has lead the way in animal phylogeography, other DNA sequences are used, most commonly chloroplast (cp) in plants and non-coding nuclear (nc) regions in both animals and plants. MtDNA has a relatively fast rate of nucleotide divergence, well suited to examining events over the last few million years, but those of cpDNA and ncDNA are an order of magnitude lower and consequently less useful for such young divergences. More slowly evolving sequences are required for deeper phylogenetic history. For more recent events, like the last 10 thousand years, highly variable markers are needed, such as microsatellites and AFLPs, but whilst useful for population studies they suffer from homoplasy and produce equivocal genealogies .Techniques for obtaining DNA sequence information are still advancing rapidly, with whole genome sequences being produced in a growing number of organisms. This allows sequences and markers to be identified and developed for many types of investigation, and some will be useful for phylogeographic studies. In particular, it is clear that genealogical data is required from several independent nuclear loci to provide a fuller and more reliable history of the species . Single The advance in DNA technology is producing a wealth of data for individuals, populations and species, and there are concomitant developments in analytical methods to divine demographic history and evolutionary relationships, and to test their significance. This progress in analysis is facilitated by access to increasingly powerful desktop computers on which the increasingly sophisticated software can be used. Haplotype sequences of a particular DNA region can be ordered into a genealogical tree or network, and hence produce their phylogeny. When combined with their population frequency and geographic distribution, this provides a strong basis for inferences on the evolutionary history of the populations and species. The usual phylogeographic approach is to build a phylogeny from haplotype sequences using distance, parsimony and maximum likelihood methods and then represent the lineages geographically. There are several approaches that are regularly used, such as DNA Distance Phylogeography, Nested Clade Analysis, Haplotype Networks, Sequence Mismatch Distribution and Genetic and Demographic Simulation, e.g. PAUP and GeoDis -10. ThisEach DNA sequence has its own genealogy and they may evolve at different rates. Furthermore, the various methods of analysis probe different aspects of the molecular and spatial history. Consequently, to reconstruct a species phylogeographic history one would ideally like to use a range of sequences  and apply a suite of pertinent analyses. This is not easy and often not possible with resources available. However, technological advances for molecules and computers have been explosive in the past decade, making much more detailed analysis possible today than only a few years ago, and this looks set to continue.The very different field of paleoclimatology is also experiencing great advances. The results of these are most pertinent to phylogeographic explanations, since they reveal the past environmental conditions and changes that have molded the evolutionary processes producing the present genetic structure. They provide a framework in which the phylogeny may be reconstructed. Past conditions can be deduced from carbon and oxygen isotopes, radiolarian skeletons, pollen grains and other residues from the sea bed, lake bottoms and ice sheets . Novel iThese major changes in distributions of species occurred latitudinally as the ice sheets advanced and retreated, altitudinally in major mountain regions, and also longitudinally where new dispersal routes became available, as for example the Bering land bridge produced by the lowered sea level. The demographic fluctuations and adaptive challenges produced by such range changes would have had both stochastic and selective effects on the genetic variation and architecture, and the consequences of these can be studied by genetic and phylogeographic approaches. Thus the once distinct fields of paleobiology and phylogeography are now being combined, and have much to tell us about how present biodiversity was structured.With some effort it is now possible to obtain DNA data from specimens across the present range of a species, but fossil data is often more limited or absent. The most useful fossil data are for Europe and North America, which have extensive networks of pollen cores; a few span 3 ice ages (400 kyr), several reach the last interglacial 125 kyr), and a larger number cover back to the last glacial maximum (LGM 23-18 kyr) . There a kyr, andDuring the LGM the ice sheets and permafrost extended towards lower latitudes, so that generally species distributions were compressed toward the equator. Boreal species survived south of the ice in North America and Europe, but large areas of the north eastern Palearctic and Beringia remained ice free and some cold-hardy species appear to have survived here. Temperate species survived further south where habitats occurred to which they were each adapted. In Europe the disjunct southern peninsulas of Iberia, Italy and Balkans were particularly important, while in North America many temperate locations occurred around 40°N between the East and West coasts. Nearer the equator the pollen record is not extensive, but conditions were generally drier in the LGM and Tropical habitats were reduced while desert and savanna increased.As a consequence, the habitats of many Boreal, Temperate and Tropical species were reduced and fragmented and they survived in refugia; but for some their habitats expanded, like those in the tundra and savanna. As the climate warmed after the LGM and the ice retreated, many Boreal and Temperate species were able to expand their ranges, as were some Tropical species. In some cases the refugial populations died out, but particularly in mountainous regions they could survive by ascending with the climate and their niche, as for example in the Alps, Andes, Appalachians and Arusha mountains. Such refugial regions allow the survival of species through several ice age cycles by ascending and descending to track their habitat, e.g. .Such events modify the genetic content and structure of populations within species, and leave some traces for which we may search. Populations, races and subspecies that have been effectively separated for several glacial cycles will show divergence through the accumulation of neutral and possibly selected DNA changes. The extent of this divergence will be proportional to the time of separation. The haplotype tree or network of an evolving DNA sequence will reflect population expansions and contractions. Increasingly these effects can be analyzed, e.g.  and placMost phylogeography has concerned Temperate biota ,26, but The phylogeographic structure, in terms of distinct regional DNA clades, is very marked in some species like the lemmings, voles and wren, moderate in the ptarmigan and dunlin, and less in the more mobile waterflea, reindeer and herring gull. The extent of DNA divergence between major clades in small mammals would suggest effective separation of up to 1 Myr, some 5–10 full glacial cycles, with further subdivision for shallower clades in more recent ice ages. In the gull, ptarmigan and reindeer the divergence among clades is low, indicating events occurring in the last or penultimate glacial cycles. Such recent structure would suggest that these species came from or were reduced to a small ancestral population in the late Pleistocene.The deeper clades of the true and collared lemmings, the root and field voles, and to some extent the shallow ones of the ptarmigan and dunlin, are remarkably parapatric and many contacts between them coincide around major features like the Urals, Lena, Kolyma and MacKenzie Rivers Fig  32]. Re. Re32]. Each distinct regional clade would have had its own refugium, but the location of these remains to be determined. There are late glacial fossils of small mammals in Central Europe and also near the southern Urals, which were both parts of the extensive tundra and could have been refugia for European and west Asian clades. An additional refugium in east Asia would account for the clades meeting in the regions of the Lena and Kolyma Rivers, while fossils for a number of species in Beringia indicate that it was probably a significant refugium . A recenThe contractions, expansions and distant colonizations involved in these late Quaternary range changes would have influenced the genetic diversity, and should have left some signs. Low haplotype diversity and shallow clades are expected when populations have been severely contracted, and the age of the subsequent expansion may be gauged by mismatch analysis. The structure of haplotype networks and nested clades also provide indications of such events. Many of the populations and clades reported show these features  dating to the Pliocene [Genetic studies in tropical rainforests of SW Amazonia and NE Australia show phylogeographic divergence that is geographically concordant across a number of taxa and also originating in the Pliocene. The first set concern some 35 species of small mammals sampled along the Jurua River, and where for the majority there is a deep phylogenetic divide coincident with the Iquitos Arch. This formed as a bulge in front of the uplifting Andes in the Pliocene creating two basins that filled with sediment. The depth of mtDNA divergence between clades in these two basins places their separation at this time . The IquPliocene . InteresGnarosophia bellendenkerensis also shows these main phylogeographic features, and with some finer subdivisions. Its distribution from the LGM to the present has been modeled using current climate envelopes for this snail species mapped onto reconstructed paleoclimate distributions [The second set of phylogeographic studies involves several birds, reptiles and frogs from the remnant strip of tropical forest in NE Queensland . This weibutions . There iibutions ,79. ThesThe phylogeographic pattern demonstrated by amphibians, reptiles and small mammals, is also found in bird species from tropical Africa and South America, which show old Pliocene lineages in the lowland forest and mixed old and recently diverged clusters in the mountains. This has lead to the proposal that such mountains provided a relatively stable environment through the ice ages and rising mountains, in which older lineages survived and new ones were created . DNA divThere have been a number of recent DNA studies of several larger mammals from Africa that provide some interesting insights into how Pleistocene climatic changes modified their ranges and hence their genetic structure and divergence Table . Whilst Connochaetes taurinus, greater kudu Tragelaphus strepsiceros and the impala Aepyceros melampus, the data support colonization northwards to the east from refugia in the south of Africa [Most phylogeographies show some 3 major clades that are associated with 3 main areas of Tropical Africa, the west, east and south, indicating that these have been major refugial areas for the development of this divergence through climatic cycles in the Late Pleistocene (Fig f Africa ,84. The Synercus caffer caffer has younger haplotypes in the south, which along with mismatch analyses suggest one or two recent colonizations from populations in eastern Africa. The DNA divergence of these from central African buffalo subspecies is perhaps only 180-130 kyr, and roughly coincident with the Cape buffalo's genetic expansion and evidence from fossils [Alcelaphus buselaphus and the topi Damaliscus lunatus probably survived in a few places in southern and eastern Africa from which they expanded with better conditions [Phacochoerus africanus is now relatively widespread, but has 3 distinct clades equivalent to subspecies, and with low divergence within each one [On the other hand, the Cape buffalo  fossils . The harnditions ,86. The each one . This poLoxodonta cyclotis and savannah L. africanus elephants mixed together in several clades [The mtDNA phylogeny of the African elephant is more complexly structured, with haplotypes of the putative species of forest l clades . It suggl clades . The cauWhilst there are common features within and distinguishing ones between the wet and dry examples reviewed from the Tropics, there are individualities to each species phylogeography; these reflect differences in biology and history that have produced differences in genetic structure. The richness and diversity seen at the species level is multiplied by that within species, and more studies are needed on biotas from such habitats to properly describe and understand them. Little is known from the species rich Tropics of SE Asia, or from the plains of S America and the large areas of Temperate Asia. There are a number of individual studies on a range of species, but one needs several representatives from each community to look for generalities. Given the species diversity in these areas, such genetic and phylogeographic knowledge is particularly important to inform sensible decisions on management and conservation of these resources.Man is in the unique position to know and predict the consequences for the environment and its biota of his innate will to survive and reproduce. Our actions are greatly modifying both of these, so we face the challenge of managing this sensibly. But we must also think of these natural and induced biotic changes in the light of future major changes in the climate. It is clear that global oscillations, producing great climatic changes, have occurred and will continue; in particular the increasingly severe Quaternary ice ages, which are now well researched and clearly demonstrated. Biotas have changed greatly due to these, and will do so again. We are currently well into an interglacial, the Holocene, and there is debate about how soon it will end and how quick this will be. If the North Atlantic conveyor is turned off, and Man may assist in this, colder conditions may return very quickly. On the other hand, in the shorter term global warming may continue, and Man may assist this! What should we do about biodiversity, and how does phylogeography inform us for this?The distribution of biodiversity across the world is largely measured as species diversity – their numbers, proportions and distinctness. But within a species there are often several geographic subspecies, and genetic studies have added greatly to knowledge of subspecific diversity, with some regions possessing more lineages and older divergence. Mountain ranges in warm Temperate and Tropical regions are seen to be important because they harbour much diversity at species, lineage and allelic levels. Phylogeographic studies reveal that this is likely a product of species surviving through climatic oscillations by tracking their habitat altitudinally and locally in a varied topography in regions not so affected by the extremes of climate change, e.g. . The souThe mountain regions in the wet Tropics of Africa and South America are very rich in species; while phylogeographic studies reveal that these contain divergences often into the Pliocene with subsequent diversification through the Pleistocene. This retention of older diversity through millions of years along with younger lineages argues that they are both generators and reservoirs of divergence and species ,80. LessBesides these more general issues, there are a number of more particular lessons and questions. For example, it has recently been noted in several butterflies that the lower genetic diversity produced by postglacial colonization of northern Europe is correlated with their recent decline, as evidenced in national records. Moreover, different deduced postglacial colonization patterns show the same correlation of low genetic diversity and population decline . This suAnother particular example is the genetic diversity created by the formation of a hybrid zone as two genomes meet with postglacial colonization, which is multiplied when several species zones coincide as a suture zone, e.g. . It has The demonstration that the extent of divergence among lineages within and between sister species generally increases from the High Arctic to the wet Tropics reflects their evolutionary age. It can be argued that the richer Tropical biotas are more valuable than the poorer temperate ones, both in terms of their present allelic, lineage and species diversity, and their long term survival. However, this overlooks the particular adaptations of Temperate species and the vast highly productive biotas they produce. The agriculture of the Temperate regions also supports much of the world's population. The consideration of regional diversity and adaptation raises a number of related questions. How well coadapted are recently assembled Temperate ecosystems? Are north Temperate and Arctic species particularly selected for colonization by repeated range change? Are genetically richer genomes more able to adapt to change? Do putative refugial regions contain genetic variation that may be useful in agriculture? And there are many more such considerations.The frequent major climatic oscillations in the last 2 My caused repeated changes in the ranges of surviving taxa, with extensive extinction and recolonization in higher latitudes and altitudinal shifts and complex refugia nearer the tropics. As a result of these past dynamics, the genetic diversity within species is highly structured spatially, with a patchwork of genomes divided by often coincident hybrid zones.The extent of divergence among lineages within and between sister species generally increases from the High Arctic to the wet Tropics and reflects their evolutionary age. Holarctic animal species show shallow but clear phylogeographic structure from the last or recent glaciations. Clades of several species are parapatric near major geographic features like rivers and mountains, suggesting they had similar range changes and refugial areas.In Temperate regions like Europe and North America there is much more diversity in the south, where it has accumulated in refugia over many ice ages, and much less in the north, where it was lost during postglacial colonization. These northern places have been colonized by species from different southern refugia, and have had little time to become closely coadapted. Furthermore, this loss of diversity in the north is implicated in the present reduction of population abundance in some species. Mammals from the Dry Tropics of Africa often show major clades in the west, east and south indicating major refugial areas for recent divergence through climatic cycles in the Late Pleistocene.Mountain ranges in warm Temperate and Tropical regions would seem to be important for the survival of lineages through climatic changes, and hence for genome divergence and speciation. Such understanding of the distribution of biodiversity carries serious implications for the theory and practice of conservation.


Drosophila males occurs by binding of two core proteins, MSL-1 and MSL-2, to a set of 35–40 X chromosome “entry sites” that serve to nucleate mature complexes, termed compensasomes, which then spread to neighboring sequences to double expression of most X-linked genes. Here we show that any piece of the X chromosome with which compensasomes are associated in wild-type displays a normal pattern of compensasome binding when inserted into an autosome, independently of the presence of an entry site. Furthermore, in chromosomal rearrangements in which a piece of X chromosome is inserted into an autosome, or a piece of autosome is translocated to the X chromosome, we do not observe spreading of compensasomes to regions of autosomes that have been juxtaposed to X chromosomal material. Taken together these results suggest that spreading is not involved in dosage compensation and that nothing distinguishes an entry site from the other X chromosome sites occupied by compensasomes beyond their relative affinities for compensasomes. We propose a new model in which the distribution of compensasomes along the X chromosome is achieved according to the hierarchical affinities of individual binding sites.It has been proposed that dosage compensation in  Drosophila is incorrect, and a new model is proposedEvidence is presented that the existing model for dosage compensation in  Drosophila males the expression of most of the genes on the single X chromosome is doubled. At least six protein-coding genes, collectively referred to as male specific lethals (msls), are required for dosage compensation , encoding an RNA helicase; males absent on the first (mof), encoding a histone acetyltransferase; and jil-1, encoding a histone kinase. The products of these genes, together with noncoding RNAs encoded by the RNA on the X genes(roX1 and roX2) (roX RNAs are thought to function in a ribonucleoprotein complex (compensasome) to mediate dosage compensation by altering chromatin structure of the male X chromosome  prevents formation of compensasomes and hence dosage compensation  and males (XY), organisms have evolved a variety of mechanisms, collectively termed dosage compensation, to equalize the levels of X-linked gene products in the two sexes. In ensation : msl-1, nd roX2) , are allnd roX2) . Recentlnd roX2) . Thus, Mromosome . In femaensation .LSP1-α) are scattered throughout the X chromosome and can reside next to dosage-compensated genes  that roughly span the size range of salivary chromosome bands seen with DNA stains . Thus, both the locations and the extents of these sites are somehow specified. Furthermore, the compensasome bands do not correspond to the bands where DNA is condensed . In addied genes . Since tmsl-1 or msl-2 mutant male  on the X chromosome, which include the roX1 and roX2 genes , indicating that the supply of compensasomes is not limiting in these circumstances. We also tested homozygous transpositions and duplications for MSL binding in males and found that we could recover MSL binding on each homozygous transposed piece (unpublished data) as well as on the X. Thus, even three copies of the same segment of the X chromosome  were able to maintain MSL binding. This result extends previous data showing that, by using specific ype male . Therefomsl-3 or mle mutant background for a subset of these transpositions  inserted at 87E17  and not an aspect of dosage compensation.The two high-affinity sites identified to date correspond to the X2 genes , and it ion site . We alsoMSLs see B. The dicis occurs on the X chromosome, we next asked if the complex could spread from the X onto an autosomal piece attached to the X by a reciprocal translocation. We tested two reciprocal translocations that interchanged large portions of the X and 3R or 2L (see roX1 (3F) and roX2 (10C) genes from one another and thus both pieces of each translocation contain a roX locus. Anti-MSL-1 staining revealed the absence of any bands on either of the 3R or 2L pieces of these translocations  and line XIII (5A) are relatively close to the roX2 (10C) and roX1 (3F) loci, respectively.To further assess if spreading in ocations , while troX transgene studies where MSL binding is observed both in the autosomal regions adjacent to the insertion site and on the wild-type autosomal homologue.We also tested two small transpositions of autosomal regions into the X C: NeitheDrosophila. It is worth noting that our experiments were a priori neutral: They could have provided compelling evidence for or against the spreading model. In both cases our results are inconsistent with the clear predictions of that model. First, we show that pieces of the X chromosome inserted into an autosome bind compensasomes in precisely the pattern characteristic of that piece of the X at its endogenous location on the X, and this property is independent of the presence of sites previously described as entry sites. Second, compensasomes do not spread from the X into autosomal pieces inserted into, or translocated onto, the X. Moreover, there is not spreading of compensasomes from autosomal insertions of pieces of the X chromosome into the autosomal regions flanking the insertion, even when such pieces contain a roX gene close to the breakpoint. These results suggest that spreading in cis is not part of the process of dosage compensation in flies. We thus propose that all of the hundreds of sites along the X chromosome where compensasomes are found in wild-type males are competent to independently recruit compensasomes.In summary, we have used chromosome rearrangements to test two central aspects of the proposed spreading model of dosage compensation in mle or msl-3 mutants qualitatively different from the other sites at which MSL bands are found in wild-type, and if so, how? Why do roX transgenes induce additional binding to adjacent autosomal sequences?Our findings raise several questions regarding previous data. Are the 35–40 sites that attract partial complexes in roX1 and roX2 genes are identified binding sites), the data are consistent with the simple view that the binding sites are homogeneous in terms of their function, but have varying affinities for compensasomes. Our finding that pieces of X chromosome transposed to autosomal locations display normal patterns of compensasome binding, irrespective of whether or not they contain high-affinity sites, removes the one functional distinction between binding sites that had been proposed. That there are not two classes of binding sites in terms of affinity for compensasomes, but rather a continuum of affinities, is strongly suggested by the recent report of mle or msl-3 mutants. Their data are consistent with a model in which compensasomes continue to bind site specifically to additional sites after all high-affinity sites are occupied, as opposed to spreading from high-affinity sites as previously proposed. Given these findings, a reasonable scenario as to how dosage compensation is achieved would be the following. As MSL expression begins, the high-affinity sites progressively sequester nascent partial or full complexes in the early stages of dosage compensation. When the amount of available complexes or its components increases, sites of higher affinity would accumulate more complexes, while low-affinity sites would remain undetectable, until the former have preferentially assembled sufficient amounts of complexes to make components available for sites with lower affinities. Thus, the compensasomes would progressively bind to different sites along the X according to the different affinities of these sites. Consistent with our model, we found that in mle or msl-3 mutants, duplications maintain binding of partial complexes at the high-affinity sites . Several features of spreading are consistent with this proposal. First, additional bands corresponding to spreading from roX transgenes contain roX RNA and the H4Lys16 modification, suggesting that they correspond to mature complexes , some of them have identified functions not related to dosage compensation, and orthologs of msl-1, -2, and -3 were not found in Caenorhabditis elegans DX, y1 w1 f1Tprb (line I); 1/C(1)DX, y1 w1 f1; Dpsn13a1/+Df(1)ct-J4, In(1)dl-49, f (line II); +72d, f1 car1/C(1)DX, y1 f1; DpbwD, bwDTpsn (line III); vco, v1 f1: in wvco/ClB, B36dTpw (line IV); +74c/FM7aTpv (line V); 64d cm1 ct6 sn3/C(1)DX, y1 w1 f1Tpw-ec, ec (line VI); +71b/FM6Tpf (line VII); 1/FM7aTpJC153, v (line VIII); 1: sssta/FM3Tpsta, sta (line IX); zh, sc1 z1 wzhTpw (line X); 2 sn3/C(1)DX, y1 w1 f1; Dpw+67k/+Df(1)w258–45, y (line XI); A/FM6Tv, v (line XII); 1.10, b1 pr1 cn1 sca1/CyOTpodd (line XIII); LtR In(2R)Cy, Cy1 amosRoi-1 cn2 sp2Df(2 l)sc19–7/In(2 l)Cy or 1 ed1 dpo2 cl1DpB19, y (line XIV); 1118; Df(3R)2–2/TM3, Sb1Dp2–2, w (line XV). Breakpoints and insertion site are referred in w; Tp/Cyo-GFP or w; Tp/TM3-GFP stocks. Non-GFP third instar male larvae were dissected for analysis. For autosome-to-X transpositions, females from lines XIV and XV were mated with w; msl2Δ3–21/CyoGFP or Dp/Y; msl2Δ3–21/CyoGFP males. Non-GFP female larvae were dissected. For mle and msl-3 mutant analysis, stocks were balanced to give p/TM3-GFPw; Tp/CyoGFP; msl3 or 12.17/CyoGFP; Tp/TM3-GFPw; prmle stocks. Females were crossed to p/CyoGFP; msl2Δ3–10/TM3-GFPw; msl3 or 1cnbw/CyoGFP; msl2Δ3–21/TM3-GFPmle males, respectively. Non-GFP third instar female larvae were dissected for salivary glands polytene chromosomes analysis. Lines expressing MSL-2 from transgenes msl2Δ3–21 and msl2Δ3–10 are described in Mle and msl-3 mutants are described in Flies were raised on standard cornmeal-yeast-agar medium. Fly stocks containing transpositions were obtained from the Bloomington Drosophila Stock Center. Their genotypes are: Glands from male third instar larvae were dissected in PBS/0.7% NaCl, prefixed in 45% acetic acid for 10 s, and then fixed for 2–3 min in lactic acid/water/acetic acid (1:2:3) solution on siliconized coverslips. Glands were squashed and coverslips flipped off after freezing the slides in liquid nitrogen. Slides were then incubated in PBS for 15 min followed by incubation with affinity-purified anti-MSL-1 antibodies (dilution 1:100) as described previously . ChromosDrosophila melanogaster BAC library  were used to map regions 3D–E, 3C, and 2D5–3A2, respectively. Specific probes were obtained from BAC clone DNA preparations using the Bionick Labelling System  according to the manufacturer's instructions. Squashes were prepared as described above. Immunostaining with affinity-purified anti-MSL-1 antibodies was followed by incubation with the appropriate biotinylated probe according to the method of Clones RP-98 17.E.2, RP-98 03.D.13, and RP-98 48.O.22 from the http://www.ncbi.nlm.nih.gov/LocusLink/) accession numbers for the genes and gene products discussed in this paper are jil-1 (LocusLink 39241), mle (LocusLink 35523), mof (LocusLink 31518), msl-1 (LocusLink 35121), msl-2 (LocusLink 33565), msl-3 (LocusLink 38779), roX1 (LocusLink 43963), roX2 (LocusLink 44673), and SXL (LocusLink 44872).The LocusLink (


L-homocysteine (Hcy) without using an intermediate methyl carrier. Although MetE displays no detectable sequence homology with cobalamin-dependent methionine synthase (MetH), both enzymes require zinc for activation and binding of Hcy. Crystallographic analyses of MetE from T. maritima reveal an unusual dual-barrel structure in which the active site lies between the tops of the two (βα)8 barrels. The fold of the N-terminal barrel confirms that it has evolved from the C-terminal polypeptide by gene duplication; comparisons of the barrels provide an intriguing example of homologous domain evolution in which binding sites are obliterated. The C-terminal barrel incorporates the zinc ion that binds and activates Hcy. The zinc-binding site in MetE is distinguished from the (Cys)3Zn site in the related enzymes, MetH and betaine–homocysteine methyltransferase, by its position in the barrel and by the metal ligands, which are histidine, cysteine, glutamate, and cysteine in the resting form of MetE. Hcy associates at the face of the metal opposite glutamate, which moves away from the zinc in the binary E·Hcy complex. The folate substrate is not intimately associated with the N-terminal barrel; instead, elements from both barrels contribute binding determinants in a binary complex in which the folate substrate is incorrectly oriented for methyl transfer. Atypical locations of the Hcy and folate sites in the C-terminal barrel presumably permit direct interaction of the substrates in a ternary complex. Structures of the binary substrate complexes imply that rearrangement of folate, perhaps accompanied by domain rearrangement, must occur before formation of a ternary complex that is competent for methyl transfer.Cobalamin-independent methionine synthase (MetE) catalyzes the transfer of a methyl group from methyltetrahydrofolate to  By solving the structure of MetE, the authors have shed light on how the chemically difficult transfer of a methyl group from methyltetrahydrofolate to homocysteine can occur Two apparently unrelated families of proteins catalyze this reaction: cobalamin-dependent methionine synthase  and cobalamin-independent methionine synthase  Organisms that synthesize or transport B12 encode the cobalamin-dependent enzyme whereas organisms that cannot obtain B12 encode only the cobalamin-independent enzyme. Escherichia coli and many other species of bacteria express both enzymes, but mammals utilize only cobalamin-dependent methionine synthase while plants and yeasts utilize only the cobalamin-independent enzyme.Methionine synthases catalyze the transfer of a methyl group from N5-methyl-5,6,7,8-tetrahydrofolate alamin·CH3-H4folate in MetH ) at 2.59 Å resolution and with Hcy at 2.20 Å resolution. The two (βα)8 barrels are formed by residues 1–351 (folate barrel) and 387–734 (Hcy barrel) and joined by an extended inter-domain linker  barrel enzymes, the active site is usually located near the C-termini of the inner barrel strands, with catalytic residues contributed by the β–α segments (loops) that join these strands to the outer helices. These barrels are topologically polar with their “tops” decorated by insertions that extend the β–α loop segments. MetE is the first example of a dual-(βα)8 barrel in which the decorated tops of the two barrels face each other to form a single active site that lies between the domains. A deep cleft between the barrels permits entry of the substrates (In (βα)bstrates A. As a rbstrates A.+2, the long β2–α2 loop begins with helix α2AH and then forms an antiparallel excursion that harbors a number of conserved residues, some of which are involved in binding folate. The β3–α3 loops both include a short helix, α3A, that carries folate-binding determinants in the Hcy  barrel. The β4–α4A segments of both barrels incorporate a conserved sequence, identified by asterisks in E. coli MetE, this sequence, Gln-Ile-Asp-Glu-Pro-Ala, is identical in both barrels.The N- and C-terminal barrels share a number of strikingly similar features that provide structural evidence for gene duplication. Both barrels incorporate long extensions in the first four β–α loops but, with the exception of α8AF B, lack iDespite the pseudosymmetry that relates the β–α loops of the two barrels, there are significant differences in the sequences and conformations of these connecting loops that distinguish the functional roles of the two barrels. The major binding determinants for the folate substrate lie primarily in the second, third, and fourth β–α loops of the C-terminal barrel. The equivalent binding site in the duplicated N-terminal barrel has been obliterated. Although the long β2–α2 hairpin of the N-terminal barrel resembles the corresponding hairpin of the C-terminal (Hcy) barrel, the potential binding groove for the folate tail is closed by extensive hydrophobic contacts between α1AF, α1F, and α2CF and by interaction with the long hairpin of the C-terminal barrel. The positioning the long β2 hairpin significantly closer to the barrel top than in the C-terminal barrel occludes the pterin-binding site B.Several other adaptations enhance the barrel–barrel interface and appear to influence the relative orientations of the barrels. The β8–α8 loop containing helix α8AF is 16 residues longer than the corresponding loop in the C-terminal domain and makes numerous inter-barrel contacts. Likewise, loop β4–α4 in the C-terminal domain is extended by six residues, increasing contacts with the N-terminal barrel. The single-domain archaeal Hcy methyltransferases that are homologous to the C-terminal domain are missing these six residues, supporting the notion that this insert evolved to enlarge the inter-domain interface.E. coli MetE had previously shown that zinc is bound by a histidine and two cysteines [T. maritima MetE. EXAFS studies of the E. coli enzyme indicated a fourth oxygen or nitrogen coordinated to zinc that seemed likely to be a water oxygen. However, the structure of zinc-replete enzyme with folate bound  enzyme and the oxidized (disulfide-bonded) form  form . CH3-H4f surface . The pte surface A. It is  surface .3-H4folate is positioned by stacking and by hydrogen bonding with conserved residues  sequence, which forms the N-terminal turn of helix α2AH. When Hcy binds, the side chain of Met468 alters its position in concert with backbone displacements of the aspartic acid and methionine residues that start the helix. Comparison of the substrate-free structure with the Hcy binary complex shows how the DMV region moves toward the zinc center when Hcy binds nsferase  and the nsferase . These hmain see A and 5. The Hcy barrels of MetE and MetH are compared in +2/Hcy site and the unusual mode of folate binding seem to have evolved to allow methyl transfer in a ternary E·Hcy·CH3-H4folate complex.Major differences in the connecting loops β1–α1 and β8–α8 displace the zinc and Hcy sites in MetE relative to MetH by approximately 6 Å so that the sulfur of Hcy is no longer on the barrel axis but is shifted toward one wall of the barrel . The alt− are made by the backbone amide and the side chain hydroxyl of Ser411, again located on the extension following β1.Despite the translation and inversion of the metal and its ligands, the orientation and local interactions of Hcy are almost identical in MetE and MetH . Both en+2/Hcy barrel, despite the fact that it is no longer a zinc-dependent thiol alkyltransferase.Curiously, it appears that distortions of the barrel strands and connecting loops need not be undone when metal binding is lost through evolution. Distortions of the barrel strands and their downstream loops are retained in the N-terminal barrel of MetE despite loss of metal and Hcy binding, and similar distortions were first observed in uroporphyrinogen decarboxylase , which iE. coli MetE at pH 7.2 are best fit to a tetrahedral ligand environment with two sulfurs (2.33 Å), one nitrogen or oxygen (2.02 Å), and one selenium (2.433 Å), in which the longer Zn–Se distance reflects the increased radius of selenium relative to sulfur [In the structure of the MetE·Hcy complex, the zinc site adopts distorted trigonal bipyramidal geometry with long bonds from the metal ion to glutamate and Hcy see . In contulfurs 2.3 Å, one Because the crystals of the Hcy binary complex of MetE were equilibrated at pH 5.2, it is possible that Hcy may be protonated in the X-ray structure. Thus, the long S–Zn bond and partial inversion at zinc might be explained by the relatively weak interaction between the thiol and the metal ion, as documented for thiol and thioether ligands in model compounds ,25,26. X− bond may be functionally important. It has been suggested that a long bond and/or distorted geometry would optimize the reactivity of zinc-dependent thiol alkyltranferases [A long Zn–Snferases  by increnferases , a five-+2 that accompanies Hcy binding to MetE is unique among the Hcy methyltransferases with known structures. There is no evidence for inversion of configuration at zinc in MetH, and inversion is precluded in BHMT, where a leucine occupies the position analogous Glu642 of MetE. Zinc motion provides a novel way to alter the distribution of charge among the zinc and its ligands, thereby modulating thiolate reactivity. The effects on the electronic structure could be larger than those resulting from the changes in bond lengths observed in other zinc-dependent alkyltransferases [The motion of Znsferases ,30. By a3-H4folate, with the pterin ring incorrectly oriented for methyl transfer. It is possible that folate binds initially in this manner to avoid blocking access to the Hcy binding site, which lies between zinc and CH3-H4folate. Space-filling models show that the Hcy-binding site remains accessible in the MetE·CH3-H4folate binary complex. The observed binding mode thus permits random addition of substrates but does not rule out a kinetically preferred order of binding.An unexpected feature of the MetE structures is the binding mode of CHE. coli MetE. Formation of the binary Hcy complex results in release of a proton to solvent . Thus Hcy is not a likely proton donor. It is more likely that an active-site residue may serve as an acid catalyst. Structures of the binary complexes do not implicate a particular residue as a general acid catalyst, but suggest possible candidates. His111 could serve as a proton donor through water if protonation were to occur before folate rearrangement. His672, part of a conserved 670Asp-Ile-His-Ser-Pro sequence, or Asp467, located in the DMV loop, may be positioned to serve as donors if protonation occurs after folate rearrangement. All three of these candidate residues are invariant in multiple sequence alignments.In both MetH and MetE, protonation of methyltetrahydrofolate is thought to occur in the ternary complexes but not in the binary folate complexes . The immE. coli MetE share a conserved Gln-Ile-Asp-Glu-Pro-Ala repeat [8 barrels, though they display characteristic distortions and can be classified into subgroups [Earlier comparisons of sequences had suggested that MetE evolved by gene duplication. The two domains of a repeat , and theubgroups , do not The idea that the zinc/Hcy barrel is the ancestral fold is based on several lines of evidence. This barrel shows significant structural homology to a broad family of zinc-dependent thiol methyltransferases, including not only MetH and BHMT but also the single-domain archaeal transferase enzymes that react with methylcobalamin. Indeed it seems likely that the three enzymes that convert Hcy to methionine, MetE, MetH, and BHMT, are all descended from a primordial zinc/Hcy barrel. In contrast, DALI searches that assess structural similarities  reveal t+2/Hcy barrel would have replicated the sites that bind Zn+2 and Hcy, but these sites have been disabled in the N-terminal barrel of contemporary MetE. Disruption of zinc and Hcy binding is effected by both residue mutations and backbone conformational changes  and cloned into pET-151D/TOPO . The expression construct contains an N-terminal leader sequence consisting of a 6X histidine tag followed by a V5 epitope, rTEV cleavage site, and residues 2–734 of the coding sequence of TM1286. This vector was overexpressed in BL21(DE3)Star (Invitrogen) by induction with 0.8 mM IPTG for 8 h in LB media supplemented with zinc sulfate. The histidine-tagged protein was purified by a 10-min 70 °C heat step, which precipitates heat-labile protein, followed by affinity chromatography on Zn(II)-NTA and elution with a 50 mM to 1.5 M glycine gradient. The protein was dialyzed against 50 mM Tris (pH 7.4) and 500 μM Tris(2-carboxylethyl)phosphine (TCEP) and concentrated to 20 mg/ml. Selenomethionine-labeled protein was expressed in M9 medium supplemented with amino acids and zinc sulfate.The TM1286 gene was PCR amplified from 1212  were grown by mixing 20 mg/ml protein 1:1 with 25% poly(ethylene glycol) 4000, 0.2 M ammonium sulfate, and 0.1 M sodium acetate (pH 4.6). Selenomethionine-labeled protein was crystallized by mixing 1:1 with 12% poly(ethylene glycol) 4000, 0.2 M ammonium sulfate, and 0.1 M sodium acetate (pH 4.6). Anapoe detergents  were used as additives and seen to have a favorable effect on crystal morphology. Crystals in a cryoprotectant solution consisting of 15% poly(ethylene glycol) 4000, 0.12 M ammonium sulfate, 0.1 M sodium acetate (pH 5.2), and 12.6%–14.1% meso-erythritol were flash-cooled in liquid nitrogen.TM1286 was crystallized by the vapor batch (microbatch) method under oil utilizing 96-well Douglas vapor batch plates and a 1:1 mixture of silicon:paraffin oil . Orthorhombic crystals of space group P23-H4PteGlu3 (a gift from Rebecca E. Taurog) for several hours. The enzyme:Hcy binary complex was formed by soaking crystals pre-equilibrated with zinc sulfate and TCEP in cryoprotective solution containing 10 mM L-Hcy (a gift from Rebecca E. Taurog) for several hours.These crystals were found to be depleted of zinc with a disulfide bond connecting the zinc ligands, Cys620 and Cys704. Zinc-replete crystals were obtained by soaking in cryoprotectant solution containing 500 μM zinc sulfate and 500 μM TCEP for 4–16 h prior to flash cooling. The enzyme:folate binary complex was formed by soaking crystals in cryoprotective solution with added 3.5 mM CHAll datasets were collected at the Advanced Photon Source (APS) at Argonne National Laboratory. Data collected at the DND-CAT beamline on a Mar225 detector were processed with XDS  whereas Experimental phases to 2.8 Å were derived from selenium SAD measurements at the selenium peak using heavy atom sites located by a three-wavelength selenium MAD experiment at lower resolution. Thirty of the 36 expected selenium sites were found and phases were determined using SOLVE version 2.05 , and sta3-H4folate complexes (the resting state of the zinc center), restraints were based on ideal tetrahedral geometry; for the Hcy complex, restraints were chosen using bond valence sums analysis [His618, 2.07 Å; Zn–SCys620, 2.31 Å; Zn–SCys704, 2.29 Å; Zn–OGlu642, 2.14 Å) gives a bond valence sum of 1.88, consistent with the known +2 oxidation state of zinc, and a net contraction of Zn–SCys bonds from 2.30 to 2.24 Å upon Hcy binding is enough to maintain the bond valence sum for the five-coordinate state with long axial bonds.In late rounds of refinement of the binary complexes, weak restraints were applied to maintain the geometry at the zinc site. For the MetE·CHanalysis  to make http://www.rcsb.org/pdb/) with accession codes 1T7L (substrate-free oxidized), 1XDJ (zinc and Hcy complex), 1XPG (zinc and methyltetrahydrofolate complex), and 1XR2 (oxidized methyltetrahydrofolate complex).Coordinates of the structures have been deposited in the Research Collaboratory for Structural Bioinformatics' Protein Data Bank (Video S1(7.9 MB WMV).Click here for additional data file.http://www.ebi.ac.uk/swissprot/) accession numbers for the gene products discussed in this paper are A. thaliana MetE (SwissProt O50008), E. coli MetE (SwissProt P25665), S. cerevisiae MetE (SwissProt P05694), T. maritima MetE/TM1286 (SwissProt Q9X112), and MetH (SwissProt P13009).The SwissProt (


Anyone who thinks plants are passive inhabitants of their environment has never seen time-lapse footage of a seedling bursting from its protective shell or a climbing vine coiling around a tree. Such films dramatize a fundamental fact of plant life: survival depends on responding to environmental cues. Shoots grow toward light and against gravity. Stems and roots curl around obstacles that block their paths.In plants, environmental cues trigger hormonal changes that in turn regulate cells' shapes and proliferation. In this way, subtle changes in the environment affect plant growth. Auxin, the first known plant hormone, spurs growth and shapes growth patterns in nearly every plant tissue throughout a plant's lifecycle. Brassinosteroids—a class of hormones chemically similar to animal steroids like testosterone—are linked to many of the same processes as auxin.Early physiological and molecular experiments gave conflicting evidence about whether auxin and brassinosteroids had similar effects. For many years, biologists believed that these hormones acted through independent signal transduction pathways—chains of molecules that relay stimuli and elicit cellular responses, such as gene expression. But in the last few years, microarray studies, which can measure the transcription of thousands of genes simultaneously, showed that auxin and brassinosteroids do regulate expression of several genes in common.PLoS Biology, Jennifer Nemhauser et al. assay the entire genome of Arabidopsis thaliana, a favorite for plant genetics studies, for effects of auxin and brassinosteroids. The group's microarray analyses show that these hormones affect transcription of about 80 genes in common—including many known players in the hormones' signal transduction pathways. To see how this regulation could occur, the research team looked at the genes turned on by both hormones to find common promoter sequences—regions of the genome that do not code for protein but instead help regulate gene transcription. They used a new computational approach to tease out promoter regions that auxin and brassinosteroid pathways both act upon, showing how these hormones have overlapping effects on gene transcription.In this issue of Arabidopsis lines. They showed that auxin and brassinosteroids greatly enhance each other's effects on stem growth, demonstrating that the interaction of these hormones is important for normal plant development. Mutants with a disabled auxin pathway don't respond normally to brassinosteroids, and vice versa. Also, mutants with abnormally high levels of auxin have a reduced number of genes that respond to brassinosteroids. Thus, these hormones act through overlapping, interdependent pathways—but they don't regulate each other directly. Instead, the researchers suggest, the pathways likely converge on the promoters of a few key genes.The group also compared the effects of auxin and brassinosteroids on seedlings' stem growth and gene expression in a variety of mutant It's still an open question why plants use these hormones with such redundant effects. Nemhauser speculates that—as is known to be the case in animals—by having dual, interdependent pathways, plants can finely tune how these ubiquitous hormones act in different cells and tissues to shape patterns of growth. By showing clearly that auxin and brassinosteroids act together and how they affect many of the same genes, Nemhauser and colleagues have set the stage for more detailed studies of how these hormones act in specific parts of plants to shape growth.


Herpes zoster oticus accounts for about 10% cases of facial palsy, which is usually unilateral and complete and full recovery occurs in only about 20% of untreated patients. Bilateral herpes zoster oticus can sometime occur in immunocompromised patients, though incidence is very rare.Diabetic male, 57 year old presented to us with bilateral facial palsy due to herpes zoster oticus. Patient was having bilateral mild to moderate sensorineural hearing loss. Patient was treated with appropriate metabolic control, anti-inflammatory drugs and intravenous acyclovir. Due to uncontrolled diabetes, glucocorticoids were not used in this patient. Significant improvement in hearing status and facial nerve functions were seen in this patient.Herpes zoster causes severe infections in diabetic patients and can be a cause of bilateral facial palsy and bilateral Ramsay Hunt syndrome. Herpes zoster in diabetic patients should be treated with appropriate metabolic control, NSAIDS and intravenous acyclovir, which we feel should be started at the earliest. Glucocorticoids should be avoided in diabetic patients. Varicella-zoster virus, member of Herpesviridae family has structural characteristics like a lipid envelope surrounding a nucleocapsid with icosahedral symmetry, a total diameter of 180–200 nm and centrally located double-stranded DNA about 125,000 bp in length. Varicella-zoster virus lies latent in sensory root ganglion for years in a patient who had chickenpox earlier. Some precipitating factor may reactivate it especially when immunity of patient wanes leading to Herpes zoster, a sporadic disease. Involvement of geniculate ganglion of sensory branch of facial nerve leads to Herpes zoster oticus also known as Ramsay Hunt syndrome. Involvement of facial nerve leads to otalgia, lower motor neuron homolateral facial paralysis and vesicular eruptions in auricle and external auditory canal. In severe cases of herpes zoster oticus, involvement of vestibulocochlear nerve leads to sensorineural hearing loss in 10% and vestibular symptoms in 40% patients. Herpes zoster oticus accounts for 10% cases of the facial palsy, paralysis is usually complete and full recovery occurs in only about 20% of untreated patients [Herpes zoster rash is characterized by unilateral vesicular eruptions with in a single dermatome. In 16% of patients of zoster, vesicles develop beyond single dermatome [Considering, rarity of bilateral herpes zoster, a diabetic patient presenting with bilateral Ramsay Hunt syndrome is being reported here.Diabetic male, 57 year old presented to us with history of pain in left ear for the last 8 days. 48–72 hrs after the onset of otalgia, patient developed facial weakness on left side along with vesicular eruptions on left conchae and in left external auditory meatus. After another 24–48 hrs patient had similar episode on right side. On the day of reporting to us patient was having bilateral facial weakness, impaired taste sensation, dryness of eyes along with decreased hearing on both sides but there was no history of vertigo or any ear discharge. There was history of stressful life events in past 6 months before the onset of rash.On examination, there was bilateral lower motor neuron facial palsy which was complete. Bell's phenomenon was present on both sides Fig-The patient was admitted and investigated. His postprandial blood sugar was 339 mg%, HbAIc was 7%. Pure tone audiometery was showing mild to moderate bilateral sensorineural hearing loss, stapedial reflexes were absent on both sides on tympanometery. There was impaired taste sensation from anterior two third of tongue. ELISA and Western Blot tests for HIV infection were negative. Liver function tests, tumor markers, thyroid hormones; serum ACE levels were all with in normal limits. Lumbar puncture revealed normal pressure. Glucose, protein and white blood cell count were all with in normal limits in CSF. Plain X-ray views of mastoid, internal auditory meatus and chest were normal. Computerized tomography of brain stem, cerebellopontine angle, temporal bone and skull base were normal. A smear from floor of vesicle stained with Giemsa stain showed degenerating cells with multiple nuclei. This favoured the clinical diagnosis of herpes zoster oticus. This diagnosis was confirmed by detection of IgM antibodies to Varicella-Zoster virus by ELISA test.Diabetes of this patient was controlled with insulin. Intravenous acyclovir was given in dose of 10 mg/kg every 8 hr for 7 days. Glucocorticoids were avoided in this patient due to diabetes, but NSAIDS were given. After 2 weeks of treatment and diabetes control, pure tone audiometery showed improvement in hearing by 10 db in all frequencies. 8 weeks later in the follow up, patient was able to close his eyes completely Fig-1c was 7.0%, so blood sugar control was not appropriate in this patient. Patient was also under severe psychological stress since last 6 months and psychological stress is identified as a potential risk factor for zoster that might operate by suppressing cell-mediated immunity [Severity and incidence of herpes zoster increases in elderly and in immunomodulated state like in AIDS, lymphoproliferative disorders, disseminated carcinomatosis, diabetes, during steroid therapy, during radio or chemotherapy . KubeyinHiroshige K et al (2002) conducted a study based on detection of varicella zoster virus DNA in tear fluid and saliva of patients with Ramsay Hunt syndrome and concluded that varicella zoster virus reactivation occurs in the unaffected side at the same level as in the affected side . This exIn the management part, as metabolic control of diabetes improves leukocyte functions and overall immune status of patient  so insulOn follow up of this patient, facial nerve recovered on both sides, recovery was more on right side. This was documented by electroneurography which showed greater amplitude of muscle compound action potentials on right side as compared to left. This might have been due to lesser time gap between the onset of acyclovir therapy and attack of zoster oticus on right side. Time gap between the onset of acyclovir therapy and attack of zoster oticus is most relevant prognostic factor in recovery of these patients and this hypothesis is supported by findings in studies of Mcgrath N. (1997) , RaschilHerpes zoster can cause severe infections in diabetic patient and can cause bilateral facial palsy and bilateral Ramsay Hunt syndrome.-Herpes zoster in diabetic patients should be treated with appropriate metabolic control, NSAIDS and intravenous acyclovir, which we feel should be started at the earliest.-Intravenous acyclovir therapy in cases of herpes zoster oticus is effective in control of disease and prevents the incidence of permanent facial palsy but treatment should be started early in the course of disease preferably with in 72 hrs from start of disease.-Glucocorticoids should be avoided in Herpes zoster patients having uncontrolled diabetes.The author(s) declare that they have no competing interests.All three authors1) Have made substantial contributions in management of this case and in conception, design, analysis and interpretation of results of this case report2) Have been involved in drafting the article or revising it critically for important intellectual content.3) Have given final approval to the version to be published.The pre-publication history for this paper can be accessed here:


Past studies suggested that tumor necrosis factor (TNF) assisted anti-tumor treatment and intensified the sensitivity of chemotherapy. However its clinical application has been curbed because of its low purity, high dosage, and strong toxicity. This research, through perspective random clinical control experiment, observed the therapeutic effect of the treatment of late malignant tumor through the injection of recombinant mutant human tumor necrosis factor (rmhTNF) combined with general chemotherapy and its adverse reactions.6u/m2 was given to the trial group, from the 1st to 7th days, the 11th to 17th days combined with chemotherapy course. The chemotherapy plan was as follows: CAP for patients with the NSCLC; FAM for patients with gastric cancer; FC for patients with colorectal cancer. One treatment cycle lasted for 21 days and two cycles were scheduled. The control group was given only the same chemotherapy as the trial group.105 patients with advanced malignant tumor were randomly divided into trial group, 69 patients, and control group, 36 patients. Injection of rmhTNF 4 × 10P = 0.022). The response rate for NSCLC in the trial group was 8/17 (47.06%), and 1/6 (16.67%) in the control group. The response rates for gastric cancer and colorectal cancer in the trial groups also were higher than those of the control groups. After the treatment the KPS is 89.00 ± 9.92 in the trial group, and 84.17 ± 8.84 in the control group, with a significant difference between the two groups (P = 0.028). The adverse reactions of rmhTNF injection included: pain in the injection area, chill, hardening and swelling and redness in the injection area, fever, ostealgia and myosalgia, and cold-like symptoms. All these adverse reactions were mild and bearable.In the trial group there was 1 CR case and 12 PR cases, and the response rate is 13/69 (18.84%); in the control group 1 PR case, the response rate 1/36 (2.78%). The response rate of the trial group was significantly higher than that of the control group (The administration of rmhTNF injection in combination with general chemotherapy is an effective and secure means in treating advanced malignant tumor. Tumor necrosis factor (TNF) is a polypeptide produced by monocytic macrophages and T-lymphocytes stimulated by endotoxin. It has P >0.05, see Table P >0.05, see Table 105 patients from our department with advanced malignant tumors diagnosed via pathologic or cytological examinations were randomly collected, of which 23 were non-small cell lung cancer, 50 gastric cancers and 32 were colorectal cancers. Moreover, 79 of them were males and 26 of them were females. The range of their ages was 25~70, with the median age 52 years old. All of them were during their later phases of either recrudescent or metastatic cancers, had no sugary indexes and were taking conservative medicine treatment. KPS scoring for them before treatment were ≥ 60, neutrophil ≥ 2.0 × 109/L, PLT ≥ 100 × 109/L and Hb ≥ 90 g/L. They had almost normal functions of the heart, liver and kidney and at least had one evaluable tumor focus. They had not taken any other anti-tumor treatment one month before the experiment and they had a prospective survival time > 3 months. All of the patients were well informed and written consents were signed. Then they were randomly divided into two groups, i.e. the trial group of 69 patients and the control group of 36 patients, the conditions of the two groups were comparable (5u/tube (number 00061) was made and provided by the Forth Military Medical University and was stored at 4°C in refrigerator.Injection of rmhTNF which was in powder form of 500 × 106u/m2 was given from the 1st to 7th days, the 11th to 17th days combined with chemotherapy course. The chemotherapy plan was as follows: CAP  for patients with the non-small cell lung cancer; FAM  for patients with gastric cancer; FC  for patients with colorectal cancer. One treatment cycle lasted for 21 days and two cycles were scheduled. For the control group, only the same chemotherapy as the trial group was given. Signs, symptoms and adverse reactions were carefully observed during the treatment. Weekly examinations of blood routine were performed before and after treatment, while liver and kidney functions, urine routine, EEG, liver ultrasonic and chest X-ray examinations were performed before and after every treatment cycle. CT examinations of the evaluable tumor focuses were performed one time before treatment, when treatments were finished and after 4 weeks of the finish.For the trial group, injection of rmhTNF 4 × 10Complete response (CR) is that, the disappearance of all lesions and no appearance of new disease for at least 4 weeks. Partial response (PR) is defined as a reduction by at least 50% in the sum of the products of the two longest diameters of all lesions maintained for at least 4 weeks with no appearance of new disease. Minimal response (MR) is different from PR with a reduction by at least 25%, but not more than 50%. Stable disease (SD) is a less than 25% reduction or less than 25% increase in the sum of the products of the two perpendicular diameters of all measured lesion with no appearance of new disease. Progression disease (PD) is that, an increase greater than 25% over the size present at entry into the study or, for patients who respond, the size at time of maximum regression, or the appearance of new areas of malignant disease. CR+PR were rated as response rate.The toxicity was followed the WHO acute and sub-acute toxic ratingP = 0.022, see Table After two treatment cycles, the trial group had 1 CR case, 12 PR cases, 11 SD cases and 18 PD cases, and the response rate was 13/69(18.84%). The control group, on the other hand, had 1 PR case, 4 MR cases, 19 SD cases and 12 PD cases, and had a response rate 1/36 (2.78%). The response rate of the trial group was significantly higher than that of the control group (P = 0.208). The response rates for gastric cancer was 12.50% (4/32), while for the controls were 0.00% (0/18) without statistic significance (P = 0.283). The response rates for colorectal cancer was 5.00% (1/20), while no response case in the controls group (0/12), but there was still no statistic significance , higher than that of the control group 1/6 (16.67%) but without statistic significance . After the treatment the KPS was 89.00 ± 9.92 in the trial group, and 84.17 ± 8.84 in the control group, with a statistic significant difference  between the two groups . No abnormalities correlated with drugs were found in liver or kidney functions, urine routine, EEG and blood pressure (see Table . No abno4N3M0IIIb". Chemotherapy of protocol CAP + rmhT NF injection (i.m.) was given from October 4 to November 14, 2001. Two weeks later, a clear relief of hard breath and cough was found. After two periods of therapy , physical examination showed shrinkage of lymph node of 0.5 × 0.5 cm above the right clavicle, and Chest CT  is a polypeptide produced by monocytic macrophages and T-lymphocytes stimulated by endotoxin. It can act as modulator to immunity and induces anti-tumor effects in hosts. It also has direct cytotoxic effects and inhibitory effects on cellular growth. It can kill the tumor cell without notable toxic effect on the normal cells . StudiesP = 0.022). Analysis of the therapeutic effects in different kind of diseases showed that the response rate for NSCLC of the trial group was 8/17 (47.06%), higher than that of the control group 1/6 (16.67%), which also means that TNFα has suppressive effects on lung adenocarcinoma[P > 0.05) between the two groups may be due to too small samples in this investigation. The response rates for gastric cancer and the colorectal cancer were both quite low, i.e. 4/32 (12.50%) and 1/20 (5.00%), respectively, while for the controls were 0/18 and 0/12, respectively (0.00%) and without statistic significance (P > 0.05). Such a result may be mainly due to the fact that most of the patients had taken combined chemotherapy before and that samples in both groups were quite small. It was thought [P = 0.028). It shows that the combined rmhTNF chemotherapy may benefit the quality of the patients' life.The present study has shown that shortly after the combined chemotherapy with rmhTNF, 24/69 (34.78%) of the focuses were more or less absorbed or subsidized, of which there were 1 CR case and 12 PR cases, with the response rate was 13/69 (18.84%), while in the control group only 5/36 (13.89%) cases had reduced focuses with only 1 PR case and the response rate 1/36 (2.78%). The response rate of the trial group was significantly higher than that of the control group (rd or 5th times, and can be relieved after taking 25 mg metacen, and can disappear automatically after the finish of the therapy. There were no significant differences between the two groups on the number of cases which showed marrow suppression or nausea / vomiting, and on the degree of those symptoms. There were no cases which showed rmhTNF-correlated abnormal liver or kidney functions, urine routine, EEG and blood pressure.It has already been reported-5,10,11 In summary, combined therapy of rmhTNF and chemotherapy has co-activating and sensitivity improving effects on the treatment of advanced malignant tumors, and may increase the recent responsive effectiveness with an improvement of the general status and quality of patients' lives. The main adverse reactions of the local injection of rmhTNF are pain in the local injection area, chill, hardening and swelling and redness in the injection area, fever, ostealgia and myosalgia and cold-like symptoms, all of which were of light to moderate degree and are tolerable.


Recent results showed that certain DEAD box protein RNA helicases, DDX3 and DDX1, play an important role in the HIV infection cycle by facilitating the export of long, singly spliced or unspliced HIV RNAs from the nucleus via the CRM1-Rev pathway. Close examination of an extensive microarray expression profiling dataset obtained from cells latently infected with HIV induced to undergo lytic viral replication indicated that additional DEAD box proteins, beyond DDX3 and DDX1, exhibit differential expression during lytic HIV replication, and in latently infected cells prior to induction into active replication. This finding provides additional evidence that the involvement of DEAD box proteins and other RNA-binding proteins may play roles in active HIV replication and in the control of viral latency. Agents targeting these functions may offer new approaches to antiretroviral therapy and the therapeutic manipulation of HIV latency. The DEAD box proteins, a family of RNA helicases containing the conserved amino acid motif Asp-Glu-Ala-Asp (D-E-A-D in the single letter amino acid code), play an essential role in many aspects of cellular RNA metabolism , inclIn a directed analysis of a large data set, which describes global changes in cellular gene patterns before and after a latently infected cell line was induced into active viral replication , we founYedavalli et al.  identifiOur work on large scale expression profiling in HIV actively and latently infected cells has been guided by the hypothesis that there is one set of cellular conditions which is ideal for normal cellular growth and homeostasis, that there is another set of conditions which may be better suited to supporting viral replication, and that HIV has evolved ways of altering the host cell so as to better support viral replication. An interesting corollary of the hypothesis is that targeting the products of the differentially expressed genes may inhibit viral replication by making the host cell environment less hospitable to viral replication . Sev, the data were subjected to statistical analyses using univariate parametric and multivariate permutation analyses, based on the one sample random variance t-statistic, where significance was based on P < 0.001 and the proportion of false discoveries was limited to 0.10 with a 90% confidence level. 1740 genes showed differential gene expression at a minimum of one timepoint during lytic replication. Hierarchical clustering analyses were performed using mAdb clustering tools, as well as Treeview . Since the data was obtained from latently infected cell lines, there may be some concern that infected primary cells may behave in a somewhat different fashion. However, the advantages of using latently infected cell lines are significant: Activation into active replication is reasonably synchronous and includes essentially all the cells, so that the signal from the cells supporting active replication is not diluted by the signal from uninfected cells or cells with virus at different stages of viral replication. Also, the signal comes only from infected cell and not from cells responding to effects from the exposure to very large numbers of defective viral particles that are in high multiplicity of infection inocula.In our study, we compared RNA samples obtained from HIV latently infected cell lines prior to induction of active replication by the integrated HIV-1 provirus and following such induction with phorbol myristyl acetate (PMA). We profiled ACH-2 cells treated with PMA side by side with similarly treated HIV-1 naïve parental A3.01 cells to assess differential cellular gene expression patterns associated with HIV lytic replication. The dataset was generated from samples obtained from three independent biological replicate experiments and at least two microarray hybridizations were done for each time point from each biological replicate (for a minimum of 6 microarrays per time point). A detailed description of the cell culture, induction, RNA isolation, and microarray labeling and hybridization methods are contained in reference . Followi[DDX10, a DEAD box protein with expression in many tissues having tumorigenic activity when fused to the nucleoporin NUP98 [DDX21, a DEAD-box protein originally identified as a nucleolar protein thought to be involved in ribosomal RNA metabolism[DDX23, a DEAD box protein first identified in U5 SnRNPs with significant homology to the yeast Prp28p splicing factor[DDX52, a human DEAD box protein identified through its homologies to a yeast gene [Figure . We founin NUP98 ,23, DDX2etabolism, DDX23, ng factor, and DDXast gene .EIF4G1 [NCBP1 (CBP80) [SMARCA2 and SMARCA5 [Other genes encoding proteins with RNA splicing/binding and RNA transport activity were also up regulated during this period, including the methylated mRNA cap binding proteins EIF4G1  and NCBP (CBP80) , which f SMARCA5 ,30. Anot SMARCA5 , indicatOur findings that several DEAD box protein genes are upregulated during HIV replication lend support to the published finding that two cellular DEAD-box proteins, including one (DDX3) that was identified in a broad-based screen for differentially expressed genes , may be In our recent publication , we showCellular genes may be differentially expressed during viral replication for many different reasons. Differential expression of cellular genes may conceivably occur because of viral actions on the host cell designed to optimize the cell for viral replication, because of cellular responses to infection aimed at inhibiting viral infection, or may be fundamentally unrelated to key aspects of viral replication. However, the findings that several DEAD box protein genes are differentially expressed during HIV replication, together with the recently published observations that two DEAD box genes, DDX3 and DDX1, exhibit differential expression during HIV replication and have important functions in HIV replication lend additional credence to the hypothesis that a careful, large scale study of differentially expressed cellular genes can provide insights into host cell factors involved in viral replication and pathogenesis. Future studies may reveal additional human co-factors for HIV replication. These cellular co-factors many represent important new therapeutic targets.The authors declare that there are no competing interests.VK designed and performed the experimental work and the data analysis. SZ directed and coordinated the study and participated in the data analysis. VK and SZ wrote the manuscript.