Literature on the spectrum of opportunistic disease in human immunodeficiency virus (HIV)-infected patients from developing countries is sparse. The objective of this study was to document the spectrum and determine the frequency of various opportunistic infections (OIs) and non-infectious opportunistic diseases, in hospitalised HIV-infected patients from north India.One hundred and thirty five consecutive, HIV-infected patients  admitted to a tertiary care hospital in north India, for the evaluation and management of an OI or HIV-related disorder between January 2000 and July 2003, were studied.Pneumocystis jiroveci pneumonia (PCP) (7.4%), cryptococcal meningitis and cerebral toxoplasmosis (3.7% each). Most of the cases of TB were disseminated (64%). Apart from other well-recognised OIs, two patients had visceral leishmaniasis. Two cases of HIV-associated lymphoma were encountered. CD4+ cell counts were done in 109 patients. Majority of the patients (82.6%) had CD4+ counts <200 cells/μL. Fifty patients (46%) had CD4+ counts <50 cells/μL. Only 50 patients (37%) received antiretroviral therapy. Twenty one patients (16%) died during hospital stay. All but one deaths were due to TB  and PCP .Fever (71%) and weight loss (65%) were the commonest presenting symptoms. Heterosexual transmission was the commonest mode of HIV-acquisition. Tuberculosis (TB) was the commonest OI (71%) followed by candidiasis (39.3%), A wide spectrum of disease, including both OIs and non-infectious opportunistic diseases, is seen in hospitalised HIV-infected patients from north India. Tuberculosis remains the most common OI and is the commonest cause of death in these patients. The first case of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) in India was detected in 1986 in the state of Tamilnadu . Even moThough the majority of HIV-infected population lives in developing nations, there is a paucity of data on natural history, pattern of disease and survival of hospitalised patients with HIV/AIDS from these regions, especially India. It is well established that manifestations of AIDS are influenced by factors such as endemic infections and malnutrition that are widely prevalent in these regions . ConventWe describe a series of 135 consecutive patients with HIV/AIDS, aged 13 years and above, admitted to the All India Institute of Medical Sciences (A.I.I.M.S.) hospital, New Delhi during the period of January 2000 through July 2003. A.I.I.M.S. is a large tertiary level teaching hospital and referral centre located in north India, attending to HIV-infected patients apart from others. The catchment area of this hospital includes the New Delhi national capital territory and neighbouring states mainly, Uttar Pradesh, Bihar, Uttharanchal, Haryana, Punjab and Himachal Pradesh. All patients were under the care of a single unit, of the three units existing in the Department of Medicine. Decision to admit was taken by the treating physician and all patients were hospitalised for the evaluation and treatment of a suspected OI or HIV-related disorder. We have earlier reported the determinants of in-hospital mortality in this series .Diagnosis of HIV infection was made using methods described previously . All pat9/L; lymphocytopenia – absolute lymphocyte count (ALC) <1.2 × 109/L; decreased CD4+ – <500 cells/μL; thrombocytopenia – platelet count <150 × 109/L; elevated erythrocyte sedimentation rate (ESR) – >20 in the first hour; hypoalbuminaemia – albumin <35 g/L; hyperglobulinaemia – globulin >40 g/L; hyperbilirubinaemia – serum total bilirubin >20.5 μmol/L; elevated liver enzymes – >2 times the upper limits of normal [aspartate aminotransferase (AST) – >100 IU/L, alanine aminotransferase (ALT) – >100 IU/L, alkaline phosphatase (ALP) – >560 IU/L]; hypoxaemia – PaO2 <12 kPa; increased arterial-alveolar oxygen gradient (A-a DO2) – >3.3 kPa, while breathing ambient air (FiO2 = 0.21).Various laboratory abnormalities were defined using cut-off values as follows: anaemia – haemoglobin <100 g/L; leucopenia – total leucocyte count (TLC) <4 × 10Mycobacterium tuberculosis in sputum culture; disseminated tuberculosis (DTB): clinical features suggestive of TB with concurrent involvement of at least two non-contiguous organs, in the presence of bacteriological and/or histopathological evidence of TB and improvement with anti-tuberculosis therapy; miliary tuberculosis (MTB): clinical presentation consistent with TB and bacteriological and/or histopathological evidence of TB and demonstration of bilateral miliary infiltrates on chest radiograph or high-resolution CT; tuberculosis meningitis (TBM): clinical and cerebrospinal fluid (CSF) features consistent with tuberculosis meningitis, with bacteriologic demonstration of M. tuberculosis in CSF or after exclusion of other aetiologies such as cryptococcus and syphilis, with evidence of tuberculosis elsewhere and/or improvement with anti-tuberculosis therapy; cryptococcal meningitis: in CSF, demonstration of Cryptococcus sp. yeast cells by India ink or antigen by latex agglutination or growth in culture; cerebral toxoplasmosis: demonstration of multiple ring-enhancing cerebral parenchymal lesions on contrast-enhanced CT or magnetic resonance imaging (MRI), in the presence of anti-toxoplasma antibody in serum and clinical response to anti-toxoplasma therapy; progressive multifocal leucoencephalopathy (PMLE): compatible clinical presentation with demonstration of characteristic cerebral white matter changes by MRI; Pneumocystis jiroveci pneumonia (PCP): bilateral, diffuse interstitial infiltrates on chest radiograph or high-resolution CT, with hypoxaemia (PaO2 <12 kPa) and sputum smears/cultures negative for aerobic bacteria and AFB and/or demonstration of Pneumocystis jiroveci in induced sputum [While genuine efforts were made to establish the diagnosis of an OI, patients at times were too sick to undergo invasive diagnostic procedures such as bronchoscopy or tissue biopsy and hence the following criteria were used to define an OI concerned. Pulmonary tuberculosis (PTB): clinical features suggestive of TB with radiological features compatible with TB on chest radiograph or computed tomographic scan (CT) and/or demonstration of acid-fast bacilli (AFB) in sputum smears or growth of d sputum .2 <9.3 kPa), corticosteroids were given in addition to oral co-trimoxazole. None of these patients received assisted ventilation. All patients were counselled and offered antiretroviral therapy, whenever indicated. Upon discharge from the hospital, patients were advised to follow-up as outpatients, on a regular and as needed basis.Patients received primary chemoprophylaxis for PCP and toxoplasmosis and received treatment for specific OIs followed by secondary prophylaxis, as per recommendations . For patEntry and analysis of all data were done using a statistical software package (SPSS for Windows, Version 10.0, SPSS Inc., Chicago, IL). Entered data were double-checked for discrepancies. Data are presented as mean ± standard deviation (SD) when distributed normally and as median with interquartile range (IQR), if the distribution was skewed. Frequency of various clinical and laboratory findings and the frequencies of individual OIs are expressed as proportions (%). The relation between ALC and CD4+ count was analysed by Pearson's product moment correlation. Paired-samples t-test was applied to assess the improvement in CD4+ cell counts following antiretroviral therapy.Over a period of about three and a half years, 135 patients with HIV/AIDS were admitted and all these patients were included in the study. Mean age of the study group was 34 ± 10 (range: 13–73) years. Females constituted less than one fourth of the study group (17%). Details of other demographic characteristics and transmission categories are presented in Table-2) and generalised lymphadenopathy was present in a considerable proportion of patients (16.3%). Poor performance on mini mental status examination (MMSE score <23) was not uncommon (20.7%). Altered sensorium and focal neurologic deficit were encountered occasionally. Anaemia was present in about half of the patients (50.5%) and among those who where anaemic, 17 (21.8%) patients were on zidovudine. A considerable number of patients were leucopenic and in 22% of patients ALC was less than 1200/μL (Table-Three symptoms namely fever, weight loss and diarrhoea were the common symptoms at presentation (70.4%, 65.2% and 23.7% respectively). Productive cough and dyspnoea were present in about a fourth of patients (Table-patients 6.3%. PooμL Table-, ten hadThe mean number of OI was 1.4 per patient. The commonest OI was TB (71.1%), followed by oral candidiasis (39.3%). Most of the cases of TB were disseminated  received antiretroviral therapy, of which three patients were taking protease inhibitor (PI) based regimens. All other patients received non-nucleoside reverse transcriptase inhibitor (NNRTI) based (nevirapine – 46 patients, efavirenz – one patient) triple-drug regimens. Follow-up CD4+ counts were done in 16 patients after three to six months of antiretroviral therapy. The mean CD4+ count increased to 224 ± 178 cells/μL from a baseline mean of 131 ± 122 cells/μL, which was statistically significant (P = 0.003). Myelosuppression during antiretroviral therapy developed in four patients, who were taking regimens containing zidovudine. One patient who was on didanosine developed acute pancreatitis Figure . AnotherTwenty one patients 15.6%) died in hospital, most of them due to TB (16 patients) and PCP (4 patients). All patients who died in hospital except for one, had CD4+ counts less than 200 cells/μL  are the major cause of morbidity and mortality in patients with HIV infection. In resource-limited settings, knowledge regarding the prevalence of various OIs might aid in making decisions regarding empirical treatment and would help to prioritise limited resources. To this end, we studied the frequency of various opportunistic conditions in this series of 135 HIV-infected patients. Nearly a half of these patients came from places other than New Delhi, being referred by medical practitioners or approached on their own. In India, HIV-infected patients are usually treated in tertiary level centres, due to lack of expertise and facilities in primary and district level hospitals. Moreover, for reasons of privacy patients and their kin prefer treatment at places far off from their home towns. This series comprises predominantly of patients from the northern states of India. Hence the findings of the present study may not apply to south India and north-east Indian states. Moreover, being a hospital based study, patients with severe illness causing rapid death and patients with milder symptoms who prefer treatment from local health facilities may not have been proportionately represented in the study group.Most of the patients were in the age group 21–40 years and males were predominantly affected. This is similar to nation-level statistics in which, of the 57781 cases of HIV/AIDS reported to the National AIDS Control Organisation (NACO), 89% of the cases were in the age group 15–44 years and 74% were males . This seThe three symptoms, namely fever, weight loss and diarrhoea were common presenting features. In this series of patients, all of whom had some opportunistic condition at presentation indicating advanced HIV disease, wasting is anticipated. The association of severe weight loss at presentation with TB in HIV-infected patients has been reported before . TB was Penicillium marneffei has been reported to be the third commonest OI in southeast Asia [P. marneffei endemicity. No case of P. marneffei infection was found in the present study. Kaposi's sarcoma, atypical mycobacterial infection and disseminated CMV disease were not seen, as has been observed by a previous study from south India [The frequencies of various OIs as observed in the present study are similar to those reported earlier from other parts of India ,9,14,15.ast Asia  and is kast Asia  which shth India . It is ath India ,24. In Ith India -27. As sth India . Apart fOverall in-hospital mortality in this series is considerable and is probably reflective of the advanced nature of disease at presentation. This is evidenced by the fact that more than 80% of patients had CD4+ counts less than 200 cells/μL and all but one deaths occurred in this group. However, as we had reported earlier, CD4+ cell counts do not determine the immediate outcome of patients with an OI, whereas the latter does . The proA wide spectrum of OIs is seen in hospitalised patients with HIV/AIDS from north India. Tuberculosis is the most common OI in this group of patients. Though uncommon, non-infectious opportunistic conditions also occur. Many patients suffer from more than one OI. Hospital mortality is significant, with TB and PCP being responsible for most of these deaths. Whenever a definite diagnosis is not established quickly, empirical treatment should be considered. Such an approach is likely to improve immediate outcome in hospitalised patients with HIV/AIDS.The author(s) declare that they have no competing interests.SKS conceived of the study, designed and coordinated the study and revised the manuscript critically for important intellectual content. TK participated in the design of the study and statistical analysis and drafted the manuscript. AB participated in the design of the study, performed the statistical analysis and revised the manuscript for important intellectual content. TG participated in the design of the study, collected the clinical data and contributed significantly to the content of the manuscript. IB participated in the design of the study, collected the clinical data and participated in drafting the manuscript. PKS participated in the design of the study and coordination and also contributed significantly to the content of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Plasmodium cause malaria, Plasmodium falciparum is the deadliest. Harbored in mosquito saliva, the parasite infects its human host as the mosquito feeds on the victim's blood.Every year, malaria kills as many as 2.5 million people. Of these deaths, 90% occur in sub-Saharan Africa, and most are children. While four species of the single-celled organism P. falciparum strains show resistance to antimalarial drugs. To develop new drugs and vaccines that disable the parasite, researchers need a better understanding of the regulatory mechanisms that drive the malarial life cycle. Joseph DeRisi and colleagues now report significant progress toward this goal by providing the first comprehensive molecular analysis of a key phase of the parasite's life cycle.Efforts to control the disease have taken on an increased sense of urgency, as more P. falciparum is a single-celled eukaryotic (nucleated) organism, it leads a fairly complicated life, assuming one form in the mosquito, another when it invades the human liver, and still another in human red blood cells (erythrocytes). The intraerythrocytic developmental cycle (IDC) is the stage of the P. falciparum lifecycle associated with the clinical symptoms of malaria. Using data from the recently sequenced P. falciparum genome, the researchers have tracked the expression of all of the parasite's genes during the IDC.While The pattern of gene expression  during the IDC is strikingly simple. Its continuous and clock-like progression of gene activation is reminiscent of much simple life forms—such as a virus or phage—while unprecedented for a free living organism. Virus and phage behave like a “just-in-time” assembly line: components are made only as needed, and only in the amount that is needed. In this respect, malaria resembles a glorified virus.P. falciparum that encode proteins of unknown function.Given the remarkable coupling of the timing of gene activation with gene function, as shown in this paper, this understanding could help identify the biological function of the 60% of genes in P. falciparum appears to be ultra-streamlined and exquisitely tuned to perform a single job: consume, replicate, and invade. The simple program regulating the life of P. falciparum may hold the key to its downfall as any perturbation of the regulatory program will likely have dire consequences for the parasite. This offers renewed hope for the design of inhibitory drugs targeted at the regulatory machinery that would irreparably foul the parasite's regulatory program, ultimately resulting in its death.


RNA amplification is required for incorporating laser-capture microdissection techniques into microarray assays. However, standard oligonucleotide microarrays contain sense-strand probes, so traditional T7 amplification schemes producing anti-sense RNA are not appropriate for hybridization when combined with conventional reverse transcription labeling methods. We wished to assess the accuracy of a new sense-strand RNA amplification method by comparing ratios between two samples using quantitative real-time PCR (qPCR), mimicking a two-color microarray assay.We performed our validation using qPCR. Three samples of rat brain RNA and three samples of rat liver RNA were amplified using several kits . Results were assessed by comparing the liver/brain ratio for 192 mRNAs before and after amplification. In general, all kits produced strong correlations with unamplified RNAs. The SenseAmp kit produced the highest correlation, and was also able to amplify a partially degraded sample accurately.We have validated an optimized sense-strand RNA amplification method for use in comparative studies such as two-color microarrays. One of the principal complications in microarray analysis of gene expression is the relatively large amount of input RNA required for each assay. On average, 1–20 μg of total RNA are required per study using glass microarrays . This is+ mRNA is reverse-transcribed and converted into double stranded cDNA using an oligo(dT) primer containing a promoter for T7 RNA polymerase. The second strand cDNA serves as a transcription template for amplified antisense RNA (aRNA) production. cDNA microarray studies using T7 amplified RNA have shown that the technique yields reproducible results that correlate with the results obtained from using total RNA [Most RNA amplification methods are based on the T7-based antisense RNA amplification technique first described by Van Gelder and Eberwine in 1990 [otal RNA ,9,11. Thsense-strand oligonucleotide probes arrayed onto a suitable substrate. Each probe sequence is designed to hybridize to a specific antisense cDNA reverse transcribed from a given mRNA species. The advantage of spotted oligo microarrays over cDNA microarrays is that the oligos can be designed to be more specific, with similar hybridization kinetics, lower homology among related transcript probes, and selection among different splice variants of the same gene. However, aRNA prepared from Eberwine-amplified mRNA would produce a sense-strand cDNA target that would not hybridize with sense-strand oligo probes on the microarray.Spotted oligo microarrays consist of 'long' 50–80 mer The Genisphere SenseAmp linear mRNA amplification method produces sense-strand amplified mRNA by incorporating a double stranded T7 promoter into the 3' end of the first strand cDNA, driving transcription of an amplified RNA with the same strandedness as mRNA. SenseAmp linear amplification also allows for the use of dT and random primers in the synthesis of cDNA. This variation on the amplification protocol may be as effective on partially degraded mRNA or, using random primers in a first-strand reaction, on RNAs lacking a poly(A) tail. Further, the use of random primers combined with dT priming may help to reduce the 3' bias associated with Eberwine-based amplification methods ,13 by imMost studies evaluating amplification validity compare unamplified to amplified material ,14-17. HThroughout the course of this study, several pre-production versions of SenseAmp were evaluated with the aim of judging the optimal method. Total RNA from replicate rat brain and liver samples was amplified using one of several different techniques including three versions of the SenseAmp method, MessageAmp from Ambion, Nugen's Ovation RNA-based single primer isothermal amplification (Ribo-SPIA) method, and as an additional study, SenseAmp amplification of partially degraded RNA samples. The ratio of amplified RNAs obtained from each method was compared via relative qPCR to that of unamplified mRNAs from the same pool to determine how accurately the relative abundances were preserved. The use of qPCR provides a much broader range of results than possible with microarrays . Relativt relative quantification method for qPCR. This method is used to compare the expression of a given gene in one sample relative to a second, and is based on the amplification efficiency of the PCR primer pairs used [The fidelity of the amplification methods was determined using the ΔΔCirs used . Quantitirs used ,11,18-21irs used , and easirs used . EquatioRatio of gene expression = ECt -ΔΔ    (1)Through comparison of the relative gene expression across the various different amplification techniques, we were able to determine that each amplification method produces amplified RNA in quantities that accurately reflect the original mRNA proportions. The SenseAmp kit exhibited the best correlation to the unamplified control, and was effective in amplifying degraded RNA samples as well. In addition, we inadvertently identified a potential bias that can arise with the use of the oligo dT in reverse transcription priming.We compared liver/brain expression ratios for a broad collection n = 192) of mRNAs before and after amplification. Rat brain and liver RNAs were chosen since we needed to begin with large quantities of unamplified materials in order to test several amplification reactions on the same starting material, and to reliably assay the unamplified RNA. Several variations on the amplification method were tested to determine which method best replicated the distribution of liver/brain ratios found in unamplified RNAs. As anticipated, each of the amplification techniques produced amplified RNA that reproduced the full range of relative quantities (RQ) between liver and brain RNAs . This high correlation using degraded RNA appeared to be due to the presence of random primer in the amplification reaction. Comparing the rank order of mRNA abundances in liver . Furthermore, single stranded DNA templates downstream of the ds T7 promoter have been shown to be very efficient T7 polymerase templates ,24 demonPrevious studies have cautioned against comparing samples using different reverse transcription primers . PrimingOur experimental design called for the use of oligo dT primer in most of the RNA amplification reactions but random primer in the cDNA synthesis phase of qPCR. This design was used for each of the comparison experiments and therefore any bias introduced by the oligo dT primed reaction would be repeated for each of the amplification techniques. The effect of the oligo dT priming in the RNA amplification was identified when the degraded RNA sample was amplified using a mixture of oligo dT primer and random primer. The result showed that the degraded sample amplification resulted in a higher correlation to the unamplified control than any of the amplification techniques including the SenseAmp amplification of intact RNA. It is our interpretation that this high correlation of the amplified, degraded samples to the unamplified qPCR samples may be due to the common use of random primers for both data sets.Overcoming the problem of tissue heterogeneity with LCM and other, similar techniques will allow the research community to focus its efforts on the biologically relevant cell types. The use of RNA amplification with these small, cell-type specific techniques provides reliable and reproducible quantities of mRNA suitable for high-throughput gene expression profiling. Amplification from small amounts of LCM-selected samples provides stronger hybridization signal and reduced biological noise attributed to the presence of other cell types.RNA amplification has been shown here, and elsewhere, to be both a useful and consistent technique for production of practical amounts of RNA when limited starting material is available. While there are several reliable amplification methods available, most amplify an antisense RNA which is suitable for cDNA microarray analysis. The most apparent benefit of the SenseAmp method is the amplification of the sense mRNA strand. This allows for the direct use of cDNA reverse-transcribed from amplified RNA as a hybridization target for oligo microarrays, and any other analysis that requires a sense-strand orientation. In addition, we observed similar liver/brain ratios between amplified RNAs and unamplified RNAs. This comparison models the relative expression ratios observed with two-color microarrays. While each of the methods tested produced acceptable results, the SenseAmp methods provided optimal correlation between unamplified samples and sense-strand amplified RNA.m between 58°C and 60°C and with an optimal length of 20 nt. The %GC content was held between 20% and 80% with no 3' GC clamp. The target amplicon for each sequence was designed to be between 50 and 150 nt with an optimal Tm of 85°C. The target mRNAs represented a broad range of sizes  and base composition . Amplicons were distributed between 5'UTR (8.7%), coding sequence (82.0%), and 3'UTR (9.2%). Primers were purchased from Sigma-Genosys (The Woodlands, TX). The final working concentration for each of the primer pairs was 50 nM. A table of target sequences and primers is available in the supplemental materials .A subset of 192 sequence targets was chosen from the Compugen/Sigma-Genosys Rat 8 K oligo library for qPCR analysis. Using previously-analyzed microarray results as a guide (not shown), we selected targets with a broad range of expression ratios from brain-specific, through common, to liver-specific mRNAs. GAPDH mRNA was also selected for normalization. Primers were designed for all 193 sequences using Applied Biosystems Primer Express software v2.0 (Applied Biosystems, Foster City, CA). Primers were designed to have a TSamples of rat brain (n = 3) and rat liver (n = 3) were frozen in liquid nitrogen and ground to a coarse powder. RNA was isolated from each sample using TRIzol (Invitrogen, Carlsbad CA). After isolation, samples of the prepared RNA were further purified using a Qiagen RNeasy column . Total RNA was quantified by UV spectrophotometry and the integrity was assessed using a Bioanalyzer model 2100 . Identical total RNA samples were divided among the 8 different experiments (including unamplified control).Degraded RNA was prepared by treatment at 65°C for 15 minutes in fragmentation buffer (40 mM Tris acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate). Samples of RNA degraded under these conditions were analyzed on an Agilent Bioanalyzer using RNA Nano chips and 2100 Expert software. Degraded samples typically had little or no 28S rRNA peak remaining and a broad smear below a weaker 18S rRNA peak, corresponding to an RNA Integrity Number (RIN) of 4.9 (using baseline correction) out of a score of 10 for RNA of ideal quality.Each of the three replicates for each tissue was amplified using the Ambion messageAmp (Ambion, Austin TX), NuGen Ovation AminoAllyl amplification (NuGen Technologies, San Carlos CA), or SenseAmp version 1, version2, or version 1–2 kits. SenseAmp, version 1–2, is the commercial version of the Genisphere SenseAmp kit. Amplification with each method was done according to the protocol outlined for each method. For each of the MessageAmp and SenseAmp amplifications, 0.75 μg of input total RNA was used. For SenseAmp with random priming, 250 ng of input total RNA was used. The final yield of amplified RNA for each method was ~36 μg. For the NuGen Ovation amplification, 70 ng of input total RNA was used to yield ~10 μg of amplified cDNA.24V] as described in the SenseAmp manual . For the degraded RNA samples, random 9 mers were added to the reverse transcription reaction at twice the mass of the input total RNA . Superscript II (Invitrogen) was used for all reverse transcriptions. The cDNA was purified using a MinElute PCR Purification Kit (Qiagen).For all versions of SenseAmp, total RNA was reverse transcribed using 100 ng of an anchored dT primer [d(T)E. coli DNA polymerase I (Invitrogen) at 16°C for 2 hours as described by the manufacturer. Double-stranded cDNA was purified using the MinElute PCR Purification Kit and used for in vitro transcription using the MegaScript kit (Ambion). Amplified sense RNA was purified using the RNeasy Kit (Qiagen) and the manufacturer's recommendation for RNA clean up.The purified cDNA was 3' tailed with dATP. A T7-promoter/oligo d(T) primer was used to initiate second strand cDNA synthesis using 10 overhang on the 3' end prehybridized in 6X ligation buffer (Roche Applied Science, Indianapolis IN). Excess unligated ds T7 promoter was removed using the MinElute PCR Purification Kit. The purified cDNA, which contained a double-stranded T7 promoter linked to a single stranded cDNA template [in vitro transcription using the MegaScript kit (Ambion). Amplified sense RNA was purified using the RNeasy Kit (Qiagen).The purified cDNA was poly d(T) tailed as described in the SenseAmp (Genisphere) product manual. Excess double-stranded T7-promoter was ligated to the 3' poly d(T) tail on the cDNA in 1X ligation buffer (Roche) at room temperature for 30 minutes. The double stranded (ds)T7 promoter consisted of equal molar amounts of a T7 promoter oligo hybridized to a complementary oligo have a 10 base d(A)template ,24, was in vitro transcription using the MegaScript kit (Ambion). Amplified sense RNA was purified using the RNeasy Kit (Qiagen) and the manufacturer's recommendation for RNA clean up.The complete process is described in the Gensiphere SenseAmp product manual. Briefly, the purified cDNA was poly d(T) tailed. A T7-promoter/oligo dA template strand with a 3' blocking group was hybridized to the poly d(T) tail of the purified cDNA. Double-stranded T7-promoter was synthesized at room temperature for 30 minutes using Klenow fragment of DNA Polymerase I (Invitrogen). The 3' blocker was used to prevent the synthesis of complete second strand cDNA during the T7 promoter "fill-in" reaction. Excess T7 promoter template was removed using the MinElute PCR Purification Kit. The purified cDNA, which contained a double-stranded T7 promoter linked to a single stranded cDNA template, was used for Five μg of each amplified RNA sample and unamplified control (with the exception of the NuGen Ovation amplification which yielded cDNA directly) was reverse transcribed into cDNA. RNA was added to 1 μl of 50 ng/μl random hexamer primer, 1 μl 10 mM dNTP mix (Sigma, St. Louis MO), and RNase-free water to make 12 μl. The mixture was denatured at 65°C for 5 min and immediately chilled. Reaction buffer and SuperScript II (Invitrogen) was then added and the volume was adjusted to 20 μl. The mixture was then incubated at 25°C for 10 minutes, 42°C for 50 minutes, and finally 70°C for 15 minutes to stop the reaction.t comparable to that derived from the 1:10 dilution in the unamplified control. cDNAs for the NuGen, 2 rounds of SenseAmp version 1–2, and SenseAmp version 1–2 on degraded RNA were all diluted 100X. cDNAs from SenseAmp version 1 were diluted 120X. The remaining version 2, version 1–2, and messageAmp cDNA samples were diluted 200X. 2 μl of diluted cDNA was added to the primer pair mix and SYBR Green Master Mix (Applied Biosystems) in each well. qPCR was conducted on the Prism 7900HT Sequence Detection System (Applied Biosystems). Plates were run for 40 cycles and fluorescence intensity measured after every cycle. For each target sequence the average cycle number at which fluorescence was detected above background in the exponential phase of amplification was obtained for the brain and liver samples. This value, Ct, or cycle number at threshold, was used for calculations of relative abundance of mRNA molecules in the liver samples compared to the brain samples for each of the amplification methods.Each treatment assay was conducted across a total of four 384-well plates per assay. Each plate targeted 48 different genes for PCR amplification, each with 3 brain and 3 liver samples as well as 6 GAPDH wells for normalization across plates. A calibrator plate was used for each treatment to determine the concentration of cDNA required from each amplification technique to produce a GAPDH Cslope of the linear regression of the normalized log fluorescence values within the "window of linearity" for each well. As per the recommendations for this calculation, PCR primer pairs with strongly deviating PCR efficiencies or correlation coefficients below 0.999 were discarded [The efficiency of each of the 192 target gene PCR primer pairs was calculated using the LinRegPCR software . Normaliiscarded  Average t qPCR analysis method, an average cycle number was determined at which fluorescence crossed a threshold above background. The resulting Ct value was recorded for each tissue type and target gene. This Ct value was normalized across plates by subtraction of the Ct value from the housekeeping gene GAPDH. This value represents the ΔCt. The ΔCt from the reference brain samples was subtracted from the ΔCt obtained from the liver samples. This gave the change in ΔCt between the two tissues or ΔΔCt. With the addition of calculated primer pair efficiencies, the ratio of gene expression for each target mRNA sequence between the two tissues was determined using equation (1). Ratios of expression values for liver tissues relative to brain (RQ) for each amplification technique were plotted in Excel (Microsoft, Redmond WA) and Pearson correlations to the unamplified control were determined using Analyze-It , an Excel Statistics Add-on.The following algorithm was applied to the results from each amplification method as well as the unamplified control. As per the ΔΔCLAG performed all qPCR assays, selected and designed primer pairs, developed databases holding all data, and prepared the first draft of the manuscript. JB, JS, KH and RCG participated in the design of the study, developed the SenseAmp reactions, and modified the reactions in response to qPCR validation results. RCG supervised SenseAmp development. RPH conceived the study, supervised the qPCR assays, and coordinated work. All authors read and approved the final manuscript.


These revertants carry either a compensatory frameshift mutation or a greater than 20-fold amplification of the leaky lac allele. A crucial distinction between alternative hypotheses for the mechanisms of adaptive mutation hinges on whether these amplification and frameshift mutation events are distinct, or whether amplification is a molecular intermediate, producing an intermediate cell type, in colonies on a pathway to frameshift mutation. The latter model allows the evolutionarily conservative idea of increased mutations (per cell) without increased mutation rate (by virtue of extra gene copies per cell), whereas the former requires an increase in mutation rate, potentially accelerating evolution. To resolve these models, we probed early events leading to rare adaptive mutations and report several results that show that amplification is not the precursor to frameshift mutation but rather is an independent adaptive outcome. (i) Using new high-resolution selection methods and stringent analysis of all cells in very young (micro)colonies (500–10,000 cells), we find that most mutant colonies contain no detectable lac-amplified cells, in contrast with previous reports. (ii) Analysis of nascent colonies, as young as the two-cell stage, revealed mutant Lac+ cells with no lac-amplified cells present. (iii) Stringent colony-fate experiments show that microcolonies of lac-amplified cells grow to form visible colonies of lac-amplified, not mutant, cells. (iv) Mutant cells do not overgrow lac-amplified cells in microcolonies fast enough to mask the lac-amplified cells. (v) lac-amplified cells are not SOS-induced, as was proposed to explain elevated mutation in a sequential model. (vi) Amplification, and not frameshift mutation, requires DNA polymerase I, demonstrating that mutation is separable from amplification, and also illuminating the amplification mechanism. We conclude that amplification and mutation are independent outcomes of adaptive genetic change. We suggest that the availability of alternative pathways for genetic/evolutionary adaptation and clonal expansion under stress may be exploited during processes ranging from the evolution of drug resistance to cancer progression.“Adaptive mutation” denotes a collection of processes in which cells respond to growth-limiting environments by producing compensatory mutants that grow well, apparently violating fundamental principles of evolution. In a well-studied model, starvation of stationary-phase  Cells can respond to stress by apparently increasing their mutation rate. This study provides evidence that there is more than one pathway by which cells achieve such a response Escherichia coli (lac genes on an F′ conjugative plasmid are starved on solid medium with lactose as the sole carbon source, selecting Lac+ revertants. Lac+ clones appear over time during selection in a population of cells that shows no net growth (i.e., is in stationary phase). In most clones, Lac+ is conferred by a compensatory −1 frameshift mutation in the lac gene (Lac+ point mutants). Lac+ point mutation during stationary phase differs from mutation during rapid growth in that it requires the recombination proteins RecA, RecBC, RuvA, RuvB, and RuvC  do not readily yield Lac+ point mutants when resubjected to selection on lactose medium, led us to propose that amplification reflects a pathway of genetic change wholly or partly separate from point mutation . Lacome Lac+ . This inome Lac+ . In keepome Lac+ . These d+ point mutants is proposed to result from transient hypermutation as part of a stress response, one consequence of which is acceleration of genetic change  . We also report the first genetic requirement specific to the adaptive amplification pathway, Pol I.lac-amplified clones grown on rich medium with the dye 5-bromo-4-chloro-3-indoyl β-D-galactoside  (e.g., lac region (+ point mutation (Experiments described below make use of the different colony color phenotypes of point-mutant and l) e.g.,   e.g., . We use l) e.g., B and 1C.c region . Sectoric region C may refmutation A.lac amplification , unstable sectoring (lac-amplified), or both. A significant problem in interpreting data from such analyses is that the picked microcolony carries with it many Lac− background cells from the selection plate . Second, because some sectored colonies are not unstable (e.g., mixtures of stable, white Lac− background cells, and blue colony formers that overlapped), all sectored colonies found were retested by plating a sample on the same X-gal antibiotic medium to determine whether there was continued instability indicative of amplification (lac-amplified cells (colony-forming units [cfu]) it should contain   and, second, by imposing stringent methods to reduce contamination of microcolonies with neighboring cells and microcolonies from the selection plate . First, fication E. Third, contain , and bec sample) Table 1.−2–10−3 in almost all microcolonies . In general, we looked at 50 to 100 cells from these replated sectored colonies, but, for three of them, we screened 3,000–4,000 cells with the same result: we could find no unstable cfu within them. In contrast, cells from lac-amplified colonies, upon replating, again give rise to sectored colonies. Therefore, most of the sectored colonies that we found give no evidence of carrying amplification of the lac locus. Sectoring in these colonies may occur by chance juxtaposition of Lac+ point-mutant cells with non-mutated background cells (which are numerous) during the plating  Those unstable cfu might be clonally related to the rest of the microcolony, or might result from chance juxtaposition of two different clones carrying the same antibiotic resistance marker. To determine the frequency at which such chance juxtapositions are expected, we analyzed the cells carried on the agar plugs on which microcolonies were harvested. We did this by plating the microcolony suspensions on medium containing an antibiotic to which cells of the microcolony were sensitive, to ascertain the frequency of marked Lac+ cells that are unrelated to a microcolony but get harvested with it. The microcolony plugs contained from 900 to 7,000 antibiotic-resistant lac− background cells each. Of eleven such plugs analyzed, one contained three unrelated (different antibiotic resistance) point-mutant Lac+ cfu, one contained one unrelated unstable cfu, and three contained unrelated cfu of the very pale sectored phenotype described above. The presence on agar plugs of cells giving rise to sectored colonies that are unrelated to the microcolony, but occur at a frequency comparable to the frequency of unstables in a microcolony of the same antibiotic-resistance genotype . Another possibility is that they contain multiple F′ plasmids (lac-amplified colonies shows that they do not advance to a Lac+ phenotype efficiently (so are not obvious precursors to point mutation).The very pale sectored colonies that we found were examined further and were shown to be unstable upon retesting but contained no cells capable of forming colonies on lactose medium under the conditions of an adaptive mutation experiment. Presumably they contain too few copies of the plasmids . Their flac-amplified and point-mutant cells, as reported by + point mutants in the colony.Thus, colonies composed of a mixture of lac-amplified cells in point-mutant microcolonies, we devised a genetic selection capable of revealing single lac-amplified cells among numerous non-amplified cells. The selection depends on the quantitative relationship between the number of copies of the chloramphenicol acetyl transferase gene (cat) and the concentration of the antibiotic chloramphenicol that cells resist  could grow on rich Luria-Bertani-Herskowitz medium (LBH) containing 100 μg/ml chloramphenicol, whereas Lac+ point-mutant cells, and those amplified at lac but not at cat, did not form colonies at this concentration. We determined that 61% or more of all lac-amplified cells are detected by this selection (As an independent test for the presence of rare s resist . We inseelection .+ point-mutant microcolonies isolated from days 4, 5, and 6 of an adaptive mutation experiment, three (4%) were found to contain a few chloramphenicol-resistant (CamR) cfu . As discussed above, this contrasts with the report of + microcolonies contain lac-amplified cells. Also, these data verify independently that the high levels of sectored, but not unstable, cfu found among microcolonies (see lac-amplification bearers, because most lac-amplified cfu would have scored positively in this selection.The data are shown in amR) cfu . All of n above;  shows thnies see  were notlac-amplified and showed CamR. The similarity of this proportion to that for lac-amplified cells in point-mutant microcolonies suggests that none of the mixtures detected represents an occurrence of lac-amplified cells that arose in the same cell clone as the point mutation. Thus, we conclude that few, if any, point-mutant microcolonies include lac-amplified cells.A negative control was performed by plating samples of cells from agar plugs that did not carry a microcolony visible on a dissecting microscope. Five of 70 such samples yielded one or a few cells that were + colonies as young as the two-cell stage and up to about 40 cells. The sequential amplification–mutagenesis model demands that all colonies as young as 2–40 cells must consist only of lac-amplified cells, because these are proposed not to generate Lac+ point mutations until the number of lac copies reaches 107 or more . This frequency of events is compatible with the observation of 30–60 Lac+ colonies per 108 cells per day, which we see routinely in adaptive mutation experiments  detected as CamR. The cell numbers ranged from one to 16 cells . Two amplification events gave Lac+ cells that were chloramphenicol sensitive, detected on lactose-minimal medium plates. Many more amplification events were detected by CamR selection than by growth on lactose medium , implying that the CamR test is more sensitive, and capable of detecting levels of amplification that are too low to allow colony formation on lactose medium.Twelve samples gave + cells (cfu), ranging in number from one to 20 cells from half the sample (implying colonies ranging in size from 2–40 cells). None of these eight contained amplified cells, as determined by CamR, or by rescoring the lactose plates after 7 d, by which time any pre-existing lac-amplified cells would have formed colonies . This model predicts that lac-amplified microcolonies of approximately 105 cells should precede point-mutant colonies. As shown in 5 cells, show a very distinct pattern that conflicts with this expectation. Only 5% of microcolonies isolated on day 3 consisted of lac-amplified cells (the rest were stable point mutant). The proportion of unstable microcolonies increases on later days. This is the pattern of arising of lac-amplified visible colonies, which are rare in the first few days of an experiment and increase in frequency later (lac-amplified cells 1 to 4 d later (lac-amplified microcolonies produce lac-amplified (not point-mutant) visible colonies.If point-mutant colonies arose from cy later . The pro d later , as expelac-amplified cells . We overcame this problem by scoring the same colonies as microcolonies and found that these small lac-amplified colonies do not grow to become point mutant  allele incubated at semi-permissive temperature, because polA null mutation causes inviability in this strain background (Harris 1997). In repeat experiments, we saw a 2- to 6-fold reduction in amplification rate. In the same experiments, adaptive mutation was unchanged or increased by up to 8-fold.If adaptive amplification were a prerequisite for point mutation, then all genetic requirements for amplification should also be required for point mutation. We report the first genetic requirement found for amplification that is not also required for point mutation: Pol I. Pol I, encoded by fication A, but adA mutant C. We use+ colonies are unable to grow to visibility in the time-span of an adaptive mutation experiment is ruled out by reconstruction experiments, in which three independent lac-amplified isolates carrying polA(Ts) or +polA formed visible colonies in 5.25 ± 0.17 d and 5.39 ± 0.24 d, respectively (mean of three strains ± standard error of the mean [SEM]). Another possibility that we considered is that the polA mutation, which causes an increase in SOS induction (lexA confers low viability in combination with polA(Ts) (data not shown). However we tested this possibility using dinB-defective cells. DinB/Pol IV accounts for most, if not all, of the requirement of adaptive mutation for SOS induction (dinB polA(Ts) show the reduction in point mutation (dinB (polA(Ts). This demonstrates that the decrease in lac-amplified colonies in polA(Ts) strains is not due to conversion/channeling of lac-amplified cells into point mutants, but rather is caused by reduction of the number of lac-amplified cells formed. We conclude that Pol I is required specifically for adaptive amplification and not point mutation. These data are incompatible with models in which amplification is a prerequisite for point mutation, and support models in which point mutation is an independent mechanism.The possibility that amplification occurs in the Pol I–deficient strain but that the Lacnduction , might cnduction . The resmutation C expecteon (dinB  and the on (dinB B reporte+ adaptive point mutants (sulA promoter (see sulA is one of about 40 SOS (DNA damage inducible) genes in E. coli (+ point-mutant and lac-amplified cultures, or between cultures grown with (lactose medium) or without (glycerol medium) selection for lactose utilization. Control experiments showed that 3 h after exposure to 20 J/m2 of ultraviolet light, all cells were induced for SOS (data not shown). Thus, amplification per se does not induce an SOS response.A final aspect of the sequential model of  mutants , Hendric mutants  suggest  mutants  is a conoter see E and 1F. E. coli . Figure lac-amplified, and screened other cells from the microcolonies for GFP production. The data presented in lac-amplified microcolonies in the distribution of cells producing GFP (p = 0.8 by Student's t-test) and that neither shows persistent SOS induction. Thus, even under adaptive mutation experimental conditions, amplification is not sufficient to induce the SOS regulon. The results also show that SOS induction does not persist for many generations after stress has been released. The data are compatible with hypermutation models for adaptive mutation  X-gal rifampicin (rif) plates to determine which colonies were point mutant and which mutation , whereinlac amplification and point mutation are alternative end points, either of which allows growth on lactose. These data are reviewed conveniently by contrast with the sequential, amplification–mutagenesis model, as follows.The experiments reported here were designed to determine whether DNA amplification is an intermediate in the formation of adaptive point mutations in the Lac system, or whether the two are independent end points of genetic change under stress, each of which adapts cells to their environment. All of the data presented support the idea that E. coli Lac system  lac-amplified cells. This model derives most of its support from the observation of sectored-colony-forming cells in colonies of point-mutant cells (The experiments reported test the amplification–mutagenesis model for the mechanism of adaptive mutation in the c system . The modc system . In the nt cells .+ colonies are not mixtures of stable point-mutant cells with 10−2–10−3 unstable (amplification bearing) cells, but rather are pure  or the same one (a duplication), but either way it is required for error-prone repair leading to mutation, and its counter-selection would decrease point mutation, even though amplification is not an intermediate in the process.An additional result previously interpreted as support for sequential models was that when a gene counter-selectable at more than one copy was placed next to ecreased . Howeverlac amplification in E. coli (lac-amplified cells (− cells starved on the same plates, we infer that only a subpopulation of the starving cells experiences this transient hypermutation (lac-specific) genetic change in response to stress, is compatible with Darwinism, which also encompasses changing rates of heritable variations  stable, alternative outcome  mutations in other genes efficiently—and by the same recombination-protein- and DinB-dependent mechanism as Lac+ adaptive point mutation—both in the F′ that carries lac (E. coli Lac assay (Unlike the hypermutable state model of ries lac  and the ries lac . A reporac assay .S)  . RpoS upS) , which mS) . HoweverS) . RpoS is, SMR3598 (FC40 zff3139::Tn10Kan), SMR533 , and PJH223, a spontaneous streptomycin-resistant mutant of SMR4562, are resistant to 10 μg/ml tetracycline, 30 μg/ml kanamycin, 25 μg/ml chloramphenicol, and 100 μg/ml streptomycin, respectively. SMR4481 was made by transposition of Tn10dTet from lambda NK1323  carrying zff3139::Tn10Kan, respectively. SMR4562 codA::cat (PJH220) was constructed by linear replacement into SMR4562[pKD46] (plasmid from cat gene from pKRP10 (fadAB3165::Tn10Kan (SMR3490) and FC40 polA12(Ts)fadAB3165::Tn10Kan (SMR3491) were constructed by P1 transduction of FC36 (fadAB3165::Tn10Kan from CAG18495 (fadAB3165::Tn10Kan from CAG18495 linked with polA12(Ts) (polA(Ts)fadAB3165::Tn10KandinB10 and the UV-resistant isogenic control strain PJH310 (FC40 fadAB3165::Tn10KandinB10). polA mutant strains were grown at 30 °C, and experiments were carried out at 37 °C (a semi-permissive temperature). SMR6039 is a derivative of SMR4562 carrying Δattλ::PsulAΩgfp-mut2: the gfp-mut2 reporter gene   with lyster gene  under thgfp-mut2 gene , as per  cells were mixed with 3% each of three or four different derivative testers that carried antibiotic-resistance markers see . MicrocoH plates  containiplug see . The celplug see A, indicaplug see B, indicad colony . The col− tester cells. The number of these that form colonies during the analysis is reduced by use of antibiotics (as described above) because most cells are not resistant, but the background of tester cells may still be equal in number to those in the microcolony  of the suspension was spread on LBH X-gal rif medium to determine cell number and which microcolonies were point mutant. Point-mutant microcolonies were then plated to select CamR on LBH plates containing X-gal, rif, and 100 μg/ml chloramphenicol. Plates were scored after 15 to 20 h at 37 °C.lac amplification (blue and white sectoring) directly on that plate. All CamR colonies were seen to be lac-amplified.To analyze background cells without microcolonies, plugs of agar were taken from areas of the plates where no microcolony was visible at 20× magnification and the cells suspended in 200 μl of buffer. Half of each suspension was plated on chloramphenicol-containing medium as above, and the other half was plated with scavenger cells onto lactose-minimal medium. Chloramphenicol plates were scored between 15 and 18 h, and lactose plates were scored at 2 d, and again at 7 d. Colonies appearing on lactose medium were scored for amplification by plating a sample of the cells on LBH X-gal rif medium. Colonies appearing on chloramphenicol-containing medium were scored for lac-amplified cells detectable by CamR selection was determined as follows. There are two reasons why lac amplification might fail to be detected with the CamR selection: first, because some lac amplifications have not co-amplified the neighboring codA::cat gene, and, second, because some cat-amplified microcolonies obtained from the lactose plates (grown selectively for Lac+ and not for CamR) might have too few cat copies for growth on 100 μg/ml chloramphenicol. We determined each of these separately. We found that 59 of 77 lac-amplified microcolonies were CamR (at least 77% had co-amplified cat), and that 84% of cells from CamR (cat-amplified) isolates grown on lactose without chloramphenicol were then able to form colonies on chloramphenicol medium. The cells that did not form colonies on chloramphenicol presumably had lost or reduced their amplification of cat and lac. This would indicate that 77% of 84% (65%) of all lac-amplified cells should be detected by the CamR selection. An independent determination of the number of cat-amplified cells that grow on chloramphenicol after growth on lactose was obtained in a reconstruction experiment in which a few CamR cells were plated with a 104-fold excess of Lac+ point-mutant cells, mimicking more closely the conditions of the experiments in which microcolonies are obtained. A total of 79% of the cat-amplified cells formed colonies on chloramphenicol medium, so 77% of 79% (61%) of all lac-amplified cells should be detected. The selection for CamR is actually a more sensitive assay for lac amplification than is selection for Lac+, as shown by the fact that only 60% of cells from the cultures that showed CamR were able to form colonies on lactose medium under the conditions of an adaptive mutation experiment, though all carriedlac amplification (indicating that 40% had too few lac copies for growth on lactose), whereas 79% were detected on chloramphenicol medium.The fraction of Microcolonies were touched with the point of a needle, and cells were transferred to LBH X-gal rif plates, where they were spread by streaking for scoring. Control experiments (not shown) showed that this procedure removed, on average, 5% of the microcolony from the plate. About 50 separate colonies from each microcolony were scored for sectoring. The remainder of the colony was left for 1 to 4 d to form a visible colony in situ. The visible colony was then sampled, plated, and scored for sectoring .+ point-mutant and lac-amplified cells were grown (separately) to saturation, then mixed in various proportions, and 0.5 μl of the mixed cell suspensions was put onto the surface of prescavenged lactose plates and grown into visible colonies. These “pseudocolonies” were suspended, and cells were spread on LBH X-gal rif plates at 100 to 200 cells per plate and scored for sectoring blue phenotypes after 2 d growth.M9 lactose liquid cultures of Lachttp://www.ebi.ac.uk/swissprot/) and Ecogene ( http://bmb.med.miami.edu/EcoGene/EcoWeb/) accession numbers for the genes and gene products discussed in this paper are cat (Swiss-Prot P62577), codA (Swiss-Prot P25524), DNA Pol I (Swiss-Prot P00582), DNA Pol IV/dinB (Swiss-Prot Q47155), fadAB (Swiss-Prot P21177), malB (Swiss-Prot P02943), gfp-mut2 (Swiss-Prot P42212), lac (Swiss-Prot P00722 and P03023), recA (Swiss-Prot P03017), recBC (Swiss-Prot P08394 and EcoGene EG10825), rpoS (Swiss-Prot P13445), ruvA (Ecogene EG10923), ruvB (Ecogene EG10924), ruvC (Ecogene EG10925), and sulA (Swiss-Prot P08846).The Swiss-Prot (


In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid-phase uptake, whereas its downregulation inhibits these processes. In polarised epithelial cells, this function is primarily restricted to the apical membrane. Rabankyrin-5 localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells, and its overexpression in polarised Madin-Darby canine kidney cells stimulates apical but not basolateral, non-clathrin-mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)pinocytosis, our studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, its active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells.The small GTPase Rab5 is a key regulator of clathrin-mediated endocytosis. On early endosomes, within a spatially restricted domain enriched in phosphatydilinositol-3-phosphate [PI(3)P], Rab5 coordinates a complex network of effectors that functionally cooperate in membrane tethering, fusion, and organelle motility. Here we discovered a novel PI(3)P-binding Rab5 effector, Rabankyrin-5, which localises to early endosomes and stimulates their fusion activity Rabankyrin-5, which is activated by the small GTPase Rab5, coordinates two disparate methods that cells use to take in solids and fluids, playing a key role in both endosome fusion and macropinocytosis In mammalian cells multiple mechanisms of endocytosis operate within a single cell to perform nutrient uptake, cellular homeostasis, neurotransmission, signal transduction, antigen presentation, and defence against pathogens. Endocytosis comprises two major categories, phagocytosis and pinocytosis, depending on the uptake of particles or fluid, respectively (reviewed in Given the dependence on actin remodelling, it is no surprise that Rho GTPases , ARF6 R, and typRab5 binding and ankyrin repeats containing protein).We have previously purified a large number of Rab5 effectors by an affinity chromatography approach based on glutathione S-transferase (GST)-Rab5-GTPγS . Among the numerous proteins purified, we focused on a prominent 130-kDa protein A. The prTo investigate the function of human Rabankyrin-5, we raised antibodies against the recombinant full-length protein. By Western blotting, these antibodies detected a predominant band of 130 kDa in both bovine brain and HeLa cytosol, in the corresponding GST-Rab5-GTPγS but not GST-Rab5-GDP affinity column eluates C. EvidenAfter EEA1  and RabeWe next investigated whether Rabankyrin-5 plays a role in homotypic early endosome fusion as well as fusion of CCVs with early endosomes, similar to EEA1  and RabeSurprisingly, we noted that some peripheral structures harbouring Rabankyrin-5 overlapped with Rab5 but either poorly or not at all with EEA1 in A431 cells A, arrowsMacropinosomes have been so far defined by a combination of criteria rather than specific molecular markers. To test whether the Rabankyrin-5 vesicles fulfil the previously described requirements of macropinosomes, we determined whether they (1) are stimulated by growth factors , (2) do In NIH3T3 fibroblasts, the staining pattern of endogenous Rabankyrin-5 was similar to that of EGF-stimulated A431 cells. Rabankyrin-5 was detected on enlarged structures, predominantly at the outermost periphery and in membrane protrusions of the cell A, arrowsIn NIH3T3 cells, Rabankyrin-5–enlarged structures were labelled by an 8-min uptake of fluorescein isothiocyanate (FITC)-dextran (MW, 10.000). Consistent with their macropinocytic nature, these structures were largely depleted of transferrin see C, arrowsTo confirm that the Rabankyrin-5 structures originated from the plasma membrane through an actin-driven process, we employed time-lapse video fluorescence microscopy on NIH3T3 cells transiently coexpressing yellow fluorescent protein (YFP)-Rabankyrin-5 and cyan fluorescent protein (CFP)-β-actin. In the sequence depicted in The GTP-dependent interaction with Rab5 suggests that Rabankyrin-5 may function as a downstream effector of Rab5 in the macropinocytic pathway. Accordingly, one would expect that Rab5 localises to macropinosomes and, together with Rabankyrin-5, is required for macropinocytosis. We found that Rab5 indeed colocalises with Rabankyrin-5 on macropinosomes see , (2) of Whereas the aforementioned results demonstrate a general requirement for Rab5 and Rabankyrin-5 in pinocytosis, they do not discriminate between clathrin-dependent and-independent endocytosis. To distinguish between the two pathways, we compared the effects of Rabankyrin-5 overexpression on transferrin and horseradish peroxidase (HRP) uptake in NIH3T3 cells, biochemically. A recombinant adenovirus was generated to overexpress Rabankyrin-5 in these cells. In comparison with mock-infected cells, Rabankyrin-5 overexpression increased the uptake of HRP, particularly after longer (10 min) incubation times A. In conTo determine whether Rabankyrin-5 is also required for macropinocytosis, we attempted to ablate the protein by RNA interference using endoribonuclease-prepared small interfering RNA (esiRNA) . Since wTo confirm the requirement of Rabankyrin-5 for fluid-phase endocytosis we used an independent method: quantification of the uptake of fluorescent dextran in NIH3T3 cells microinjected with affinity-purified anti–Rabankyrin-5 antibodies. Following microinjection, FITC-dextran uptake was internalised for 30 min to label macropinosomes. Microinjection of anti–Rabankyrin-5 antibodies inhibited FITC-dextran uptake by 53% ± 5% when compared with cells injected with preimmune serum , whereasApical pinocytosis in polarised epithelial cells and growth factor-induced macropinocytosis share mechanistic properties, like dependence on actin  and PI3-This was confirmed and extended by immunogold electron microscopy on ultrathin frozen sections of mouse proximal tubule. Rabankyrin-5 showed weak labelling of the apical microvilli-covered surface of proximal tubule cells but labelled large electron-lucent structures underlying the apical surface A and 9B.To functionally address the role of Rabankyrin-5 in pinocytosis, we used filter-grown Madin-Darby canine kidney (MDCK) cells as an established system to study polarised trafficking. Alexa 488 dextran was internalised for 13 min from either the apical or the basolateral chamber. Although endogenous Rabankyrin-5 exhibited partial colocalisation with fluid-phase marker internalised from the basolateral side (unpublished data), a striking degree of overlap between the internalised tracer and Rabankyrin-5 on often ring-shaped structures underlying the apical region was observed A. These We next measured fluid-phase uptake biochemically by quantifying the internalisation of HRP either from the apical or the basolateral side for various periods of time. Filter-grown MDCK cells were either infected with adenovirus encoding Rabankyrin-5 or control virus prior to HRP internalisation. While internalisation of HRP from the basolateral plasma membrane did not significantly vary D, uptakeSince the pioneering work of Here, we provide evidence that Rabankyrin-5 is a novel Rab5 effector which, in addition to being localised to early endosomes, is associated with macropinosomes and promotes their formation according to previously established criteria. First, Rabankyrin-5 localised to enlarged vesicles that were stimulated by growth factors (e.g., EGF). Second, these vesicles differed from early endosomes by the lack of constitutively endocytosed molecules, such as transferrin, and components of the transport machinery, such as EEA1. Third, the vacuoles originated from the plasma membrane and engulfed extracellular fluid. Fourth, their formation depended on PI3-K activity, and fifth, they followed actin rearrangements at the plasma membrane.Macropinosomes are formed by the closure of membrane protrusions generated upon actin-mediated membrane ruffling. Although membrane ruffling is required for macropinocytosis, it seems not to be sufficient for macropinosome formation . Our datHow does the function of Rab5 and Rabankyrin-5 in macropinocytosis relate to early endosome trafficking? We have previously proposed a model whereby Rab5, via the recruitment of its effectors, generates and maintains a spatially restricted and functionally specialised membrane domain on the early endosomes . The RabClathrin-dependent endocytosis and macropinocytosis are independent but interconnected pathways. Whereas macropinosomes and early endosomes remain segregated in EGF-stimulated A431 cells , they caStimulation of epithelial cells with either EGF or phorbol esters increases fluid-phase uptake while reducing internalisation via clathrin-mediated endocytosis . Our resIn addition to macropinocytosis in nonpolarised cells, we have found that Rabankyrin-5 regulates apical, non-clathrin-mediated pinocytosis in polarised epithelial cells. The role of apically stimulated pinocytosis is crucial for the physiology of various organs. For example, it plays an important role in the reabsorption of proteins from the glomerular filtrate in the renal proximal tubule . RabankyIn conclusion, the identification of Rabankyrin-5 opens new opportunities for investigating the molecular principles underlying macropinocytosis and its regulation by signalling molecules. The dual role of Rab5 and Rabankyrin-5 in endosome and macropinosome function argues for a role in coordinating these two different endocytic mechanisms. Future work is required to identify the molecular partners of Rabankyrin-5 and to establish the mechanisms responsible for its membrane targeting and its role in endocytic membrane dynamics.Phospholipids were purchased from Sigma (St. Louis, Missouri, United States) and Calbiochem . FITC- or Texas Red-conjugated dextran , rhodamine-conjugated EGF, and rhodamine-conjugated transferrin were purchased from Molecular Probes (Eugene, Oregon, United States). Wortmannin and LY 294002 were from Calbiochem. A431 and NIH3T3 cells were grown in DMEM (high glucose) supplemented with 10% (v/v) heat-inactivated foetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine. MDCK II cells were grown in MEM plus 10% FCS. MDCK II cells stably transfected for FcLR 5–27  were a gThe 130-kDa protein band was excised from gels and enzymatically digested. The tandem mass spectroscopy protein sequencing procedure was performed as described previously . Bovine Recombinant proteins of Rabankyrin-5 were expressed in SF plus insect cells according to the manufacturer's instructions using Rabankyrin-5 cDNA subcloned into pfastBAC vector or subcloned into pGEX-6P series and expressed in BL21 cells.Human anti-EEA1 serum (1:10.000) was a gift from Ban Hok Toh , whereas a mouse monoclonal EEA1 antibody, a sheep polyclonal anti-HRP antibody and an antibody against phosphotyrosine (α-4G10) were purchased from BD Bioscience (Heidelberg, Germany), Abam (Cambridge, United Kingdom), and Upstate Biotechnology (Lake Placid, New York, United States), respectively. Fluorescently labelled secondary antibodies were purchased from Molecular Probes. Recombinant full-length Rabankyrin-5 was used to raise a polyclonal antibody in rabbit. Human full-length Rabankyrin-5 was cloned into pEYFP vectors containing an N-terminal tag . Recombinant adenovirus encoding full-length Rabankyrin-5 was generated according to the manufacturer's protocol (AdEasy). cDNAs encoding Rab5wt were fused to the amino termini of ECFP or EYFP, as previously described . The ant35S-Methionine–labelled proteins were transcribed and translated in vitro using a TnT-coupled transcription-translation kit (Promega, Madison, Wisconsin, United States). For Rab5 effector recruitment assays, in vitro-translated proteins were incubated with glutathione-sepharose beads complexed with GST-Rab5-GTPγS or GST-Rab5-GDP and eluted as described previously (The GST-Rab5 affinity chromatography was performed as in eviously .Cells were transfected with plasmids using FuGENE6 according to the manufacturer's instructions (Roche, Basel, Switzerland). After 16–24 h, transfected cells were washed twice with PBS and fixed with 3% paraformaldehyde and immunofluorescence labelling was performed according to standard procedures. Cells were mounted in moviol and examined on a confocal microscope  with a 100×/1.40 plan-Apochromat objective. Fluorescent images were collected using the Leica IM500 Image Manager and processed using Adobe Photoshop v5.5 .NIH3T3 cells grown on glass coverslips and transfected with plasmids encoding RN-tre were incubated with 3 mg/ml FITC-labelled dextran (MW, 70.000) in DMEM supplemented with 1% FCS and 20 mM HEPES (pH 7,4), fixed, and stained with rhodamine-conjugated antirabbit secondary antibody. Dextran uptake was determined by quantifying grey values of thresholded, fluorescent images of at least 15 cells using MetaVue 6.1r3 and ImageJ (NIH).In vitro fusion assays were performed using early endosomes labelled with biotinylated transferrin or antitransferrin antibody as well as CCVs labelled with biotinylated transferrin prepared from HeLa cells as described previously . The conTime-lapse epifluorescence video microscopy was performed using an Olympus IX70 (Olympus, Hamburg, Germany) inverted microscope equipped with a polychrome II monochromator (TILL Photonics, Martinsried, Germany), a custom filter block for simultaneous visualization of YFP and CFP , a 100× oil-immersion objective (NA 1.35, Olympus) attached to a PIFOC z-SCAN , an incubation chamber (37 °C), and a 12-bit CCD digital camera IMAGO  (TILL Photonics), controlled by TILLvisION v3.3 software (TILL Photonics). Time-lapse video sequences of YFP and CFP images were merged as RGBs using TILLvisION v3.3. They were exported as single TIFF files and either further processed using Adobe Photoshop 5.5 and Illustrator 8.0 or converted into QuickTime movies using ImageJ (NIH).Anaesthetized mice were perfusion fixed with 4% PFA, 100 mM HEPES (pH 7,25), and 0,2% sucrose either directly or 5 min after injection with 5 mg/g body weight HRP (Sigma) into the femoral vein. Kidneys were excised and postfixed 6 h at 4 °C. Some tissue pieces were cryoprotected in 2,3 M sucrose overnight at 4 °C, frozen in liquid nitrogen, and cryosectioned on a Leica UCT .2 and 1 mM MgCl2 (PBS++) containing 2 mg/ml BSA. Cell surface-bound transferrin was efficiently removed (95%) by three incubations over 10 min in IM (pH 3,4). Extracellular HRP was quenched in the cold by incubating the cells for 20 min with 20 mM MESNA in 100 mM NaCl, 50 mM Tris (pH 8,7), followed by two washes with ice-cold PBS and a further incubation for 10 min with ice-cold 50 mM iodoacetamide in PBS. For recycling experiments, cells were transferred again to 37 °C in fresh medium that was collected at the indicated time points. Thereafter, cells were washed once more with PBS and extracted for 15 min at 4 °C with 300 μl of lysis buffer (1% w/v Triton X-100, 0,1% w/v SDS, 20 mM HEPES [pH 7,4], and 100 U/ml DNase). Total HRP activity present in the cells was determined in duplicates out of at least three independent experiments using the previously described enzymatic method (MDCK cells were grown on 12-mm, 0,4-μm pore Transwell polycarbonate filters (Corning Costar, Cambridge, Massachusetts, United States). Ninety-six hours after seeding, cells were infected from the apical side for 4 h at 37 °C with adenoviruses in 250 μl of complete medium. After changing medium, cells were incubated for 18 h at 37 °C and then used for biochemical assays or microscopy. NIH3T3 and MDCK cells grown on coverslips in 24-well plates were infected in 500 μl of medium. HRP uptake was performed as described previously . In briec method  and stanc method .A431 cells were seeded in 24-well plates the day prior to transfection. Cells were transfected using oligofectamine  with either 300 ng/ml double- or single-stranded (control) esiRNA derived from the I.M.A.G.E. clone (IMAGE:258664) according to the protocol of Cells or tissues were fixed with 8% PFA in PHEM buffer and then processed for frozen sectioning according to published methods . Thawed Figure S1NIH3T3 cells were pretreated for 20 min with wortmannin (100 nM) and incubated for 25 min with 0,5 μg/ml rhodamine-labelled transferrin and 2,5 mg/ml FITC-labelled dextran (MW, 70.000), fixed, processed for immunofluorescence, and analysed by confocal scanning microscopy. Scale bar represents 10 μm.(8.4 MB EPS).Click here for additional data file.Figure S2NIH3T3 cells were either microinjected with (A and D) preimmune or (B and C) affinity-purified polyclonal α–Rabankyrin-5 antibodies and subjected to a 30-min incubation at 37 °C with (A and B) 3 mg/ml FITC-conjugated dextran (MW, 70.000) or (C and D) 1 μg/ml rhodamine-conjugated transferrin. Cells injected with immune but not preimmune antibodies showed a significant reduction of the fluid-phase marker, while the uptake of transferrin was not significantly perturbed.p > 0.001). Values shown are means ± standard deviation of at least 15 cells. Scale bars represent 10 μm.(D) Fluid-phase dextran quantification of single cells microinjected for indicated antibodies by measuring internalised fluorescence intensity ((44.0 MB EPS).Click here for additional data file.Video S1Images were taken every 3 s over a time period of 15 min. RGB stacks were converted to QuickTime format using ImageJ (NIH). The movie is played at 15 frames per second.(11.1 MB MOV).Click here for additional data file.http://www.ncbi.nlm.nih.gov/LocusLink) accession numbers of proteins discussed in this paper are H-Ras (ID 3265), v-Src (ID 6714), Arf6 (ID 382), hVps34 (ID 5289), p110β (ID 5291), Rab34 (ID 83871), Rab5 (ID 5868), Ankhzn (ID 51479), Rab4 (ID 5867), Rab7 (ID 7879), Rab11 (ID 8766), Rabenosyn-5 (ID 64145), EEA1 (ID 8411), Caveolin-1 (ID 857), RN-tre (ID 9712), Transferrin (ID 7018), EGF (ID 1950), LAMP-1 (ID 16783), Prominin-1(ID 8842), PIKfyve (ID 200576), RhoA (ID 387), and PKC-α (ID 5578).The LocusLink (


PLoS Biology, volume 2, issue 3:In Table of ContentsPage iiiThis photograph was used on the March 2004 Table of Contents, where Adam Lazarus, who generously supplied the image, should have been acknowledged. We apologize for this omission.


Blocks of duplicated genomic DNA sequence longer than 1000 base pairs are known as low copy repeats (LCRs). Identified by their sequence similarity, LCRs are abundant in the human genome, and are interesting because they may represent recent adaptive events, or potential future adaptive opportunities within the human lineage. Sequence analysis tools are needed, however, to decide whether these interpretations are likely, whether a particular set of LCRs represents nearly neutral drift creating junk DNA, or whether the appearance of LCRs reflects assembly error. Here we investigate an LCR family containing the sulfotransferase (SULT) 1A genes involved in drug metabolism, cancer, hormone regulation, and neurotransmitter biology as a first step for defining the problems that those tools must manage.Sequence analysis here identified a fourth sulfotransferase gene, which may be transcriptionally active, located on human chromosome 16. Four regions of genomic sequence containing the four human SULT1A paralogs defined a new LCR family. The stem hominoid SULT1A progenitor locus was identified by comparative genomics involving complete human and rodent genomes, and a draft chimpanzee genome. SULT1A expansion in hominoid genomes was followed by positive selection acting on specific protein sites. This episode of adaptive evolution appears to be responsible for the dopamine sulfonation function of some SULT enzymes. Each of the conclusions that this bioinformatic analysis generated using data that has uncertain reliability (such as that from the chimpanzee genome sequencing project) has been confirmed experimentally or by a "finished" chromosome 16 assembly, both of which were published after the submission of this manuscript.SULT1A genes expanded from one to four copies in hominoids during intra-chromosomal LCR duplications, including (apparently) one after the divergence of chimpanzees and humans. Thus, LCRs may provide a means for amplifying genes (and other genetic elements) that are adaptively useful. Being located on and among LCRs, however, could make the human SULT1A genes susceptible to further duplications or deletions resulting in 'genomic diseases' for some individuals. Pharmacogenomic studies of SULT1Asingle nucleotide polymorphisms, therefore, should also consider examining SULT1A copy number variability when searching for genotype-phenotype associations. The latest duplication is, however, only a substantiated hypothesis; an alternative explanation, disfavored by the majority of evidence, is that the duplication is an artifact of incorrect genome assembly. Experimental and computational results estimate that 5–10% of the human genome has recently duplicated . These eAt the same time, the appearance of LCR duplicates may be an artifact arising from one of a number of problems in the assembly of a genome of interest. Especially when classical repetitive sequences are involved, it is conceivable that mistaken assembly of sequencing contigs might create in a draft sequence of a genome a repeat where none exists. In the post-genomic world, rules have not yet become accepted in the community to decide when the burden of proof favors one interpretation (a true repeat) over another (an artifact of assembly). Again, these rules will emerge over time through the study of individual cases.Through the assembly of many case studies, more general features of duplication and evolutionary processes that retain duplicates should emerge. Although each LCR family originates from one progenitor locus, no universal features explain why the particular current progenitor loci have been duplicated instead of other genomic regions. From an evolutionary perspective, duplicated material is central to creating new function, and to speciation. One intriguing hypothesis is that genes whose duplication and recruitment have been useful to meet current Darwinian challenges find themselves in regions of the chromosome that favor the generation of LCRs.Browsing a naturally organized database of biological sequences, we identified human cytosolic sulfotransferase (SULT) 1A as a recently expanded gene family with biomedically related functions. SULT1A enzymes conjugate sulfuryl groups to hydroxyl or amino groups on exogenous substrates (sulfonation), which typically facilitates elimination of the xenobiotic by the excretory system . Sulfonap-nitrophenol and α-naphthol [Three human SULT1A genes have been reported ,13. The naphthol -19. The naphthol . High renaphthol -26. Aminnaphthol -30.SULT1A1, 1A2, and 1A3 exist in the human population [SULT1A1*2 contains a non-synonymous polymorphism, displays only ~15% of wild type sulfonation activity in platelets, and is found in ~30% of individuals in some populations [SULT1A1*2 allele is a cancer risk factor [SULT1A1*1 allele is counterbalanced by risks associated with its activation of pro-carcinogens. For example, SULT1A enzymes bioactivate the pro-carcinogen 2-amino-α-carboline found in cooked food, cigarette smoke and diesel exhaust [Polymorphic alleles of pulation -33. An aulations . Numerouk factor -36, althk factor . Ironica exhaust . The sulSULT1A3, transcriptionally active, and not found in the chimpanzee. In addition, we report that all four human SULT1A genes are located on LCRs in a region of chromosome 16 replete with other LCRs. A model of SULT1A gene family expansion in the hominoid lineage (humans and great apes) is presented, complete with date estimates of three preserved duplication events and identification of the progenitor locus. Positively selected protein sites were identified that might have been central in adapting the SULT1A3 and 1A4 enzymes to their role in sulfonating catecholamines such as dopamine and other structurally related drugs.Recently, it has become widely understood that placing a complex biomolecular system within an evolutionary model helps generate hypotheses concerning function. This process has been termed "phylogenomics" . ThroughSULT1A1 and 1A2 genes are tandemly arranged 10 kilobase pairs (kbp) apart in the pericentromeric region of chromosome 16, while the SULT1A3 gene is located ~1.7 million base pairs (Mbp) away  was located on an LCR  due to nonallelic homologous recombination between LCRs on chromosome X ,44. As dSULT1A4 gene region from the human reference genome was so similar to that of the SULT1A3 region (>99% identity) that the differences were near those that might arise from sequencing error or allelic variation. It was conceivable, therefore, that some combination of sequence error, allelic variation, and/or faulty genome construction generated the appearance of a SULT1A4 gene that does not actually exist. We therefore searched for additional evidence that the SULT1A4 gene was material.The sequence of the We asked whether any evidence was consistent with the hypothesis of an artificial SULT1A4 LCR from erroneous genome assembly, as opposed to the existence of a true duplicate region. Here, the quality of the genomic sequencing is important. The junction regions at the ends of the SULT1A4 LCR were sufficiently covered; at least nine sequencing contigs overlapped either junction boundary Figure . This amSULT1A4 possessed adenine (A) and SULT1A3 possessed guanine (G). Thus, if two genes do exist, they differ by one silent transition at the third position of codon 35. The untranslated regions, however, contained thirteen nucleotide differences while the introns contained seven additional differences  and 11% of CAG (1A3) codon 35 alleles were detected in one population. Why were the frequencies not more equal, as would be expected if SULT1A4 is always CAA and SULT1A3 is CAG? One hypothesis is that SULT1A3 is indeed CAG/CAA polymorphic as reported, while SULT1A4 is always CAA. Interestingly, in both the chimpanzee and gorilla, codon 35 of SULT1A3 is CAA. This implies that the ancestral SULT1A3 gene (prior to duplication) likely had a CAA codon. An A to G transition might have been fixed in a fraction of SULT1A3 genes after the divergence of humans and great apes. If this scenario is true, some transcripts assigned to SULT1A4 on the basis of codon 35 may actually be from individuals expressing the ancestral CAA version of SULT1A3.The codon 35 A/G polymorphism was reported as allelic variation in e et al. . It is cWe aligned the coding sequences of all available SULT1A genes and used various nucleotide distance metrics and tree-building algorithms to infer the gene tree without constraints. The unconstrained topology placed platypus as the out group, with the placental mammals ordered (ox,(pig,(dog,(rodents)),(rabbit,(primates)))). This differed from the topology inferred while constraining for the most likely relationships among mammalian orders (platypus,((dog,(ox,pig)), ((rabbit,(rodents)), primates))) . We consUsing the transition redundant exchange (TREx) molecular dating tool , we placSULT1A1 gene. Syntenic regions have conserved order of genetic elements along a chromosomal segment and evidence of synteny between homologous regions is useful for establishing relationships of orthology and paralogy. Human SULT1A1 is most like rodent Sult1a1 in sequence and function and before the advent of whole genome sequencing it was assumed that they were syntenic and therefore orthologous [SULT1A1, SULT1A2, or SULT1A4 loci but detected synteny involving the SULT1A3 loci and both rodent genomes . This finding and the TREx date estimate for the SULT1A3/1A4 duplication event at ~3 Ma indicate that SULT1A4 is a human invention not shared by chimpanzees – our closest living relatives.SULT1A3 and 1A4 LCRs were 99.1% identical overall  selection for mutant forms of the native proteins better able to contribute to fitness.A/KS) is greater than unity. Various models of evolutionary sequence change can be used to calculate these ratios. The simplest assumes a single KA/KS ratio over the entire tree (one-ratio). More complex models assume an independent ratio for each lineage (free-ratios), variable ratios for specific classes of sequence sites (site-specific), or variable ratios for specific classes of sequence sites along specified branches (branch-site specific) [Positive selection of protein function can best be hypothesized when the ratio of non-synonymous (replacement) to synonymous (silent) changes normalized to the number of non-synonymous and synonymous sites throughout the entire gene sequence (Kpecific) -57.2 distribution with degrees of freedom equal to the difference in free parameters between the models [A/KS ratio >1 and a statistically significant LRT [Estimating the free parameters in each of these models by the maximum likelihood method  enables e models . Evidenccant LRT .A/KS ratios for each branch in the 1A gene tree by maximum likelihood with the PAML program [SULT1A3/1A4 genes from the single SULT1A gene in the last common ancestor of hominoids  had KA/KS >1 , or encounter enough examples where a lower standard works, and therefore come to accept a lower standard routinely .SULT1A4 gene.Genomics has not yet accumulated enough examples for the culture to define the standards for a burden of proof. In the example discussed here, several lines of reasoning would be applied to analyze the sulfotransferase gene family. First, the fact that the draft genome for chimpanzee contains three paralogs, while the draft genome for human contains four, would normally be interpreted (as it is here) as evidence that an additional duplication occurred in the time since chimpanzee and humans diverged. It would also, however, be consistent with the loss of one of four hypothetical genes present in the common ancestor of chimpanzee and humans in the lineage leading to chimpanzee. Another possibility is that the finishing stages of the chimpanzee genome project will uncover a ca. 150 MY separates contemporary rodents from contemporary primates) that the comparison provides no information. And no closer out group taxon (e.g., orangutan) has had its genome completely sequenced.Normally, one would resolve this question using an out group taxon, a species that diverged from the lineage leading to chimpanzee and human before chimpanzee and human themselves diverged. The nearest taxa that might serve as an out group today are, however, rat and mouse. As noted above, they diverged so long ago  are distinguished (to favor post-speciation duplication) based on an analysis of the silent nucleotide substitutions using the TREx metric. The very small number of nucleotide differences separating the SULT1A3 and SULT1A4 paralogs must be the consequence of allelic polymorphism in the only gene that exists. This is indeed how some of the data were initially interpreted.This comparison, however, potentially suffers from the statistics of small numbers. The number of differences in the coding region (exactly one) is small. By considering ~10 kbp of non-coding sequence, however, additional differences were found. It is possible that in the assembly of the human genome, a mistake was made that led to the generation of a SULT1A4 region that does not actually exist. In this hypothesis, the ~20 nucleotide differences between the SULT1A3 and SULT1A4)? Or does the evidence favor the hypothesis that the SULT1A4 gene is an illusion arising from gene assembly error coupled to sequencing errors and/or allelic variation at ca. 20 sites? The culture does not yet have a standard of assigning the burden of proof here, although a choice of hypothesis based simply on the count of the number of mistakes that would need to have been made to generate each hypothesis (none for the first, at least three for the second) would favor the former over the latter. Thus, perhaps naively, the burden of proof now favors the former, and we may proceed to generate the biological hypothesis on top of the genomic hypothesis.Does the preponderance of evidence favor the hypothesis of a very recent duplication to generate a pair of paralogs  substantiate these findings.Multiple SULT1A genes were apparently useful inventions by our stem hominoid ancestor. Following the duplication of an ancestral primate SULT1A gene ~32 Ma, positive selection acted on a small proportion of sites in one of the duplicates to create the dopamine sulfonating SULT1A3 enzyme. In the example presented here, the evidence of adaptive change at certain sites is corroborated by the When studying well-characterized proteins as we have done here, episodes of functional change can be identified by piecing together several lines of evidence. It is not immediately possible, unfortunately, to assemble as much evidence for the majority of proteins in the biosphere. Thus, an important goal in bioinformatics is to recognize the signal of functional change from a restricted amount of evidence. Of the three lines of evidence employed here , structural biology, with its obvious connections to protein function and impending growth from structural genomics initiatives, will probably be the most serviceable source of information for most protein families. This should be especially true for protein families not amenable to experimental manipulation, or with deep evolutionary branches where codon-based metrics are unhelpful. If we are to exploit the incontrovertible link between structure and function, however, new structural bioinformatic tools and databases relating protein structure to sequence changes occurring on individual branches are much needed.SULT1A3 and SULT1A4 human genes should establish the existence of the two separate genes. Second, a reverse transcription-PCR experiment would be expected to uncover transcriptional activity for the SULT1A3 and SULT1A4 human genes. Since this paper was submitted, these experiments have been done, and indeed confirm our predictions made without the experimental information [This bioinformatic study makes several clear predictions. First, a PCR experiment targeted against the variation between the hypothetical ormation . Furtherormation  that conSULT1A3 coding region using the BLAST-like alignment tool [SULT1A3 locus. The other locus was dubbed SULT1A4 following conventional naming for this family. The coding sequence and genomic location of SULT1A4, as well as expressed sequences derived from SULT1A4, have been deposited with the GenBank Third Party Annotation database under accession [Genbank:BK004132].The July 2003 human reference genome (based on NCBI build 34) was queried with the ent tool , and seaent tool . Two disent tool  as the SSULT1A3 and ~500 kbp of sequence surrounding SULT1A4 were downloaded from NCBI and compared using PIPMAKER [To determine the extent of homology between the SULT1A3 and 1A4 genomic locations, ~500 kbp of sequence surrounding PIPMAKER ,70. BefoPIPMAKER .The Human Recent Segmental Duplication Page  was consThe bacterial artificial chromosome contigs supporting each member of the SULT1A LCR family, and the known genes within each LCR, were inspected with the UCSC genome browser . The DNASULT1A1 and 1A2) and [Genbank:AC145040] (SULT1A3) and chimpanzee whole genome shotgun sequences [Genbank:AACZ01082721] (SULT1A1), [Genbank:AADA01101065] (SULT1A2), and [Genbank:AACZ01241716] (SULT1A3) using PIPMAKER exon analysis. These new SULT1A genes have been deposited with the GenBank Third Party Annotation database under accession numbers [Genbank:BK004887-BK004892]. DNA sequences were aligned with CLUSTAL W [The MASTERCATALOG was used for performing initial inspections of the SULT gene family and for delivering a non-redundant collection of SULT1A genes. Additional SULT1A ORFs were extracted from gorilla working draft contigs [Genbank:AC145177] (LUSTAL W . The mulLUSTAL W . PairwisLUSTAL W  and PAUPLUSTAL W  softwareA/KS ratios, base frequencies, codon frequencies, branch lengths, and the transition/transversion bias were determined by the maximum likelihood method with the PAML v3.14 program [Parameter estimates of site class proportions, K program . Positivn) of two-fold redundant codons (Lys, Glu, Gln, Cys, Asp, Phe, His, Asn, Tyr) where the amino acid had been conserved in pairs of aligned sequences, and the number of these codons where the third position was identically conserved (c) were counted by the DARWIN bioinformatics platform [n and c values for all SULT1A genes is presented as supplementary data [see c/n quotient equals the fraction of identities (f2) in this system, or the transition redundant exchange (TREx) value [kt values) by the following equation: kt = - ln [(f2 - Eq.) / (1 - Eq.)], where k is the rate constant of nucleotide substitution, t is the time separating the two sequences, and Eq. is the equilibrium state of the TREx value [k) was estimated as 3.0 × 10 -9 changes/site/year for placental mammals (T. Li, D. Caraco, E. Gaucher, D. Liberles, M. Thomson, and S.A.B., unpublished data). These estimates were determined by sampling all pairs of mouse:rat and mouse:human orthologs in the public databases and following accepted placental mammal phylogenies and divergence times [k.Starting with aligned DNA sequences, the number (platform ,81. The data see . The c/nx) value . TREx vaEx value . The equce times ,83. TherHuman-chimpanzee genome alignments were inspected at the UCSC Genome Browser. Human-rodent genome alignments were examined with the VISTA Genome Browser ,51,84. VSULT1A3 were downloaded from NCBI UniGene [SULT1A3 or SULT1A4.All expressed sequences ascribed to  UniGene  and aligkbp (kilobase pairs); LCR (low copy repeat); Mbp (million base pairs); Ma (million years ago); ORF (open reading frame); SULT (sulfotransferase); TREx (transition redundant exchange); VISTA .M.E.B carried out the study and drafted the manuscript. S.A.B participated in designing the study and preparing the manuscript.Multiple sequence alignment of SULT1A genes.Multiple sequence alignment of SULT1A genes used in all phylogenetic analyses. Characters conserved in all sequences are indicated with asterisks.Click here for filen and c values for SULT1A genes. Pairwise n and c values between SULT1A genes. The names of the sequences are the row-headers and the column-headers. Lower triangular matrix contains n values, and upper triangular matrix contains c values.Pairwise Click here for file