Intestinal microbiota composition of interleukin-10 deficient C57BL/6J mice and susceptibility to Helicobacter hepaticus-induced colitis.

The mouse pathobiont Helicobacter hepaticus can induce typhlocolitis in interleukin-10-deficient mice, and H. hepaticus infection of immunodeficient mice is widely used as a model to study the role of pathogens and commensal bacteria in the pathogenesis of inflammatory bowel disease. C57BL/6J Il10(-/-) mice kept under specific pathogen-free conditions in two different facilities (MHH and MIT), displayed strong differences with respect to their susceptibilities to H. hepaticus-induced intestinal pathology. Mice at MIT developed robust typhlocolitis after infection with H. hepaticus, while mice at MHH developed no significant pathology after infection with the same H. hepaticus strain. We hypothesized that the intestinal microbiota might be responsible for these differences and therefore performed high resolution analysis of the intestinal microbiota composition in uninfected mice from the two facilities by deep sequencing of partial 16S rRNA amplicons. The microbiota composition differed markedly between mice from both facilities. Significant differences were also detected between two groups of MHH mice born in different years. Of the 119 operational taxonomic units (OTUs) that occurred in at least half the cecum or colon samples of at least one mouse group, 24 were only found in MIT mice, and another 13 OTUs could only be found in MHH samples. While most of the MHH-specific OTUs could only be identified to class or family level, the MIT-specific set contained OTUs identified to genus or species level, including the opportunistic pathogen, Bilophila wadsworthia. The susceptibility to H. hepaticus-induced colitis differed considerably between Il10(-/-) mice originating from the two institutions. This was associated with significant differences in microbiota composition, highlighting the importance of characterizing the intestinal microbiome when studying murine models of IBD.

Diverse gene cassettes in class 1 integrons of facultative oligotrophic bacteria of River Mahananda,West Bengal, India.

BACKGROUND: In this study a large random collection (n=2188) of facultative oligotrophic bacteria, from 90 water samples gathered in three consecutive years (2007-2009) from three different sampling sites of River Mahananda in Siliguri, West Bengal, India, were investigated for the presence of class 1 integrons and sequences of the amplification products. METHODOLOGY/PRINCIPAL FINDINGS: Replica plating method was employed for determining the antibiotic resistance profile of the randomly assorted facultative oligotrophic isolates. Genomic DNA from each isolate was analyzed by PCR for the presence of class 1 integron. Amplicons were cloned and sequenced. Numerical taxonomy and 16S rRNA gene sequence analyses were done to ascertain putative genera of the class 1 integron bearing isolates. Out of 2188 isolates, 1667 (76.19%) were antibiotic-resistant comprising of both single-antibiotic resistance (SAR) and multiple-antibiotic resistant (MAR), and 521 (23.81%) were sensitive to all twelve different antibiotics used in this study. Ninety out of 2188 isolates produced amplicon(s) of varying sizes from 0.15 to 3.45 KB. Chi-square (χ(2)) test revealed that the possession of class 1 integron in sensitive, SAR and MAR is not equally probable at the 1% level of significance. Diverse antibiotic-resistance gene cassettes, aadA1, aadA2, aadA4, aadA5, dfrA1, dfrA5, dfrA7, dfrA12, dfrA16, dfrA17, dfrA28, dfrA30, dfr-IIe, blaIMP-9, aacA4, Ac-6'-Ib, oxa1, oxa10 and arr2 were detected in 64 isolates. The novel cassettes encoding proteins unrelated to any known antibiotic resistance gene function were identified in 26 isolates. Antibiotic-sensitive isolates have a greater propensity to carry gene cassettes unrelated to known antibiotic-resistance genes. The integron-positive isolates under the class Betaproteobacteria comprised of only two genera, Comamonas and Acidovorax of family Comamonadaceae, while isolates under class Gammaproteobacteria fell under the families, Moraxellaceae, Pseudomonadaceae, Aeromonadaceae and Enterobacteriaceae. CONCLUSIONS: Oligotrophic bacteria are good sources of novel genes as well as potential reservoirs of antibiotic resistance gene casettes.

Molecular characterization of woodchuck CD4 (wCD4) and production of a depletion monoclonal antibody against wCD4.

CD4 T cells play an important role in the immune response against hepatitis B virus (HBV) infection. Woodchucks represent an excellent animal model to study HBV infection. In this study, we characterized the cDNA sequence of woodchuck CD4 (wCD4). The deduced wCD4 protein has four extracellular immunoglobulin-like domains comparable to the other mammalian CD4 molecules. The important extracellular cysteine residues and the intracellular tyrosine protein kinase-binding site of wCD4 are also conserved. The deduced wCD4 protein shows 53-63% identity with the counterparts of other mammalians. Phylogenetic analysis indicates that wCD4 is closely related with the counterparts of primates. Two polyclonal antibodies (pAbs) and four monoclonal Abs (mAbs) against wCD4 were produced. Two pAbs and one mAbs (G2) were found to effectively suppress ConA induced proliferation in vitro. Anti-wCD4 mAb G2 depleted 60% of CD4 cells from healthy woodchucks, while the remaining CD4 cells responded well to ConA stimulation. This work provides a basis for studying CD4 T cell mediated immune responses against HBV infection in the woodchuck model.

Evolution of homospermidine synthase in the convolvulaceae: a story of gene duplication, gene loss, and periods of various selection pressures.

Homospermidine synthase (HSS), the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, is known to have its origin in the duplication of a gene encoding deoxyhypusine synthase. To study the processes that followed this gene duplication event and gave rise to HSS, we identified sequences encoding HSS and deoxyhypusine synthase from various species of the Convolvulaceae. We show that HSS evolved only once in this lineage. This duplication event was followed by several losses of a functional gene copy attributable to gene loss or pseudogenization. Statistical analyses of sequence data suggest that, in those lineages in which the gene copy was successfully recruited as HSS, the gene duplication event was followed by phases of various selection pressures, including purifying selection, relaxed functional constraints, and possibly positive Darwinian selection. Site-specific mutagenesis experiments have confirmed that the substitution of sites predicted to be under positive Darwinian selection is sufficient to convert a deoxyhypusine synthase into a HSS. In addition, analyses of transcript levels have shown that HSS and deoxyhypusine synthase have also diverged with respect to their regulation. The impact of protein-protein interaction on the evolution of HSS is discussed with respect to current models of enzyme evolution.

A transcriptomic approach for exploring the molecular basis for dosha-balancing property-based classification of plants in Ayurveda.

In Ayurveda, a healthy body is defined by a balance among the three doshas (Vata, Pitta, Kapha) and ailments result due to imbalances among them. It prescribes specific plant parts/tissues collected in a season-specific manner for curing dosha-related imbalances but the plants prescribed for treating a particular dosha imbalance belong to taxonomically diverse families and often contain similar classes of phytomolecules, making it difficult to provide a phytochemical validation for any similarity that might exist among them. This exploratory study hypothesised that plants of the same dosha-curing group may have similarity at the transcript level. For proving/disproving the hypothesis, cDNA-AFLP and specific expression subset analysis (SESA) were carried out on the Ayurveda-defined active tissues of four representative plants each of the three dosha-balancing groups. cDNA-AFLP analyses indicated that even though the plants belonging to a particular dosha-group may widely differ at the transcript level, there is a small fraction of transcripts that is monomorphic among their active tissues. SESA (Tester-active tissue cDNA; Driver-cDNA from other major tissue[s]) generated 803 subtractive ESTs from the twelve plants that yielded 150 unigenes upon assembly (of ESTs from each plant separately). Cross-plant EST assembly for plants in the same dosha group also corroborated the results. Although a distinct pattern of transcripts was not observed across all the plants in a particular dosha group, some commonalities were obtained that need further characterization towards searching for the hitherto elusive similarity among plants of the same group.

Agave tequilana MADS genes show novel expression patterns in meristems, developing bulbils and floral organs.

Agave tequilana is a monocarpic perennial species that flowers after 5-8 years of vegetative growth signaling the end of the plant's life cycle. When fertilization is unsuccessful, vegetative bulbils are induced on the umbels of the inflorescence near the bracteoles from newly formed meristems. Although the regulation of inflorescence and flower development has been described in detail for monocarpic annuals and polycarpic species, little is known at the molecular level for these processes in monocarpic perennials, and few studies have been carried out on bulbils. Histological samples revealed the early induction of umbel meristems soon after the initiation of the vegetative to inflorescence transition in A. tequilana. To identify candidate genes involved in the regulation of floral induction, a search for MADS-box transcription factor ESTs was conducted using an A. tequilana transcriptome database. Seven different MIKC MADS genes classified into 6 different types were identified based on previously characterized A. thaliana and O. sativa MADS genes and sequences from non-grass monocotyledons. Quantitative real-time PCR analysis of the seven candidate MADS genes in vegetative, inflorescence, bulbil and floral tissues uncovered novel patterns of expression for some of the genes in comparison with orthologous genes characterized in other species. In situ hybridization studies using two different genes showed expression in specific tissues of vegetative meristems and floral buds. Distinct MADS gene regulatory patterns in A. tequilana may be related to the specific reproductive strategies employed by this species.

[Analysis on correlation between 3-hydroxy-3-methylglutary-coenzyme A reductase gene polymorphism of Glycyrrhiza uralensis and content of glycyrrhizic acid].

OBJECTIVE: To reveal the 3-hydroxy-3-methylglutary-coenzyme A reductase (HMGR) gene polymorphism of Glycyrrhiza uralensis, and the correlation between HMGR gene polymorphism and the content of glycyrrhizic acid. METHOD: Liquorice plants containing different content of glycyrrhizic acid were used as materials. RT-PCR was used to amplify their HMGR gene sequences, which were connected with vector pMD19-T for clone sequencing. Multiple alignments were performed to analyse HMGR gene polymorphism of G. uralensis. Then the correlation between HMGR gene polymorphism and the content of glycyrrhizic acid was revealed. RESULT: HMGR gene sequences polymorphism included codon mutation, base substitution mutation, copy number polymorphism and allele heterozygosity. There were 4 types of mutations in HMGR gene coding amino acid sequences, namely -HSL, -HSV, GALLV, GALSV. Among them, -HSV type was common in liquorice plants, -HSL type only existed in liquorice plants with low content of glycyrrhizic acid, and GALSV type only existed in liquorice plants with high content of glycyrrhizic acid. CONCLUSION: HMGR gene sequences of G. uralensis are highly polymorphic and related to the content of glycyrrhizic acid.

Cloning and characterization of cDNAs encoding for long-chain saturated acyl-ACP thioesterases from the developing seeds of Brassica juncea.

Four types of cDNAs corresponding to the fatty acyl-acyl carrier protein (ACP) thioesterase (Fat) enzyme were isolated from the developing seeds of Brassica juncea, a widely cultivated species amongst the oil-seed crops. The mature polypeptides deduced from the cDNAs showed sequence identity with the FatB class of plant thioesterases. Southern hybridization revealed the presence of at least four copies of BjFatB gene in the genome of this amphidiploid species. Western blot and RT-PCR analyses showed that the BjFatB class thioesterase is expressed poorly in flowers and leaves, but significantly in seeds at the mid-maturation stage. The enzymatic activities of different BjFatB isoforms were established upon heterologous expression of the four BjFatB CDSs in Escherichia coli K27fadD88, a mutant strain of fatty acid beta-oxidation pathway. The substrate specificity of each BjFatB isoform was determined in vivo by fatty acid profile analyses of the culture supernatant and membrane lipid of the recombinant K27fadD88 and E. coli DH10B (fadD(+)) clones, respectively. The BjFatB1 and BjFatB3 were predominantly active on C18:0-ACP substrate, whereas BjFatB2 and BjFatB4 were specific towards C18:0-ACP as well as C16:0-ACP. These novel FatB genes may find potential application in metabolic engineering of crop plants through their over-expression in seed tissues to generate stearate-rich vegetable fats/oils of commercial importance.

[Neprilysin--structure of the gene and protein product and the localization of expression]. / Neprylizyna--budowa genu i produktu bialkowego oraz lokalizacja ekspresji.

Neprilysin (NEP, CD10, CALLA - common acute lymphoblastic leukaemia antigen, neutral endopeptidase, enkephalinase) is a zinc-dependent metallopeptidase, which is involved in the metabolism of a number of regulatory peptides and plays an important role in turning off peptide signalling at the cell surface. NEP gene is located on chromosome 3q 25.1-q25.2 and is composed of 24 exons. Four types of NEP cDNAs have been identified resulting from alternative splicing of exons 1, 1bis, 2a or 2b to the common exon 3. Neprilysin is expressed in normal and malignant hematopoietic cells and in epithelial cells of many organs. In kidneys, it is expressed in podocytes, renal proximal tubular epithelium and in smooth muscles of the vessels.

Identification of salt-induced genes from Salicornia brachiata, an extreme halophyte through expressed sequence tags analysis.

Salinity severely affects plant growth and development causing crop loss worldwide. We have isolated a large number of salt-induced genes as well as unknown and hypothetical genes from Salicornia brachiata Roxb. (Amaranthaceae). This is the first description of identification of genes in response to salinity stress in this extreme halophyte plant. Salicornia accumulates salt in its pith and survives even at 2 M NaCl under field conditions. For isolating salt responsive genes, cDNA subtractive hybridization was performed between control and 500 mM NaCl treated plants. Out of the 1200 recombinant clones, 930 sequences were submitted to the NCBI database (GenBank accession: EB484528 to EB485289 and EC906125 to EC906292). 789 ESTs showed matching with different genes in NCBI database. 4.8% ESTs belonged to stress-tolerant gene category and approximately 29% ESTs showed no homology with known functional gene sequences, thus classified as unknown or hypothetical. The detection of a large number of ESTs with unknown putative function in this species makes it an interesting contribution. The 90 unknown and hypothetical genes were selected to study their differential regulation by reverse Northern analysis for identifying their role in salinity tolerance. Interestingly, both up and down regulation at 500 mM NaCl were observed (21 and 10 genes, respectively). Northern analysis of two important salt tolerant genes, ASR1 (Abscisic acid stress ripening gene) and plasma membrane H+ATPase, showed the basal level of transcripts in control condition and an increase with NaCl treatment. ASR1 gene is made full length using 5' RACE and its potential role in imparting salt tolerance is being studied.

Development of a cDNA microarray of zebra mussel (Dreissena polymorpha) foot and its use in understanding the early stage of underwater adhesion.

The underwater adhesion of the zebra mussel (Dreissena polymorpha) to substrates is a complex process that is controlled by a delicate apparatus, the byssus. As a critical activity of the byssus glands embedded in the zebra mussel feet, byssogenesis is highly active to produce numerous byssal threads from the settled juvenile stage through the adult stage in its life cycle. This lifelong activity helps the zebra mussel to firmly attach to substrata underwater, thereby causing severe economic and ecologic impacts. In an attempt to better understand the zebra mussel's byssus activity, a cDNA microarray (ZMB) including 716 genes, generated from a Suppression Subtractive Hybridization (SSH) cDNA library, was printed and used for the comparison of gene expression during zebra mussel adhesion and non-adhesion. To better understand the byssogenesis mechanism, RNA samples from the zebra mussel feet with byssogenesis and without byssogenesis were used in a two-color hybridization to reveal the gene differential expression in the two states. Based on the P values (P<0.05), Fifty-two ESTs were found as differentially expressed genes and were divided into two groups, upregulated and downregulated groups according to there logFC values. With the false discovery rate (FDR) adjustment, seven were identified from the upregulated group and nine from the downregulated group. Phylogenetic analysis indicated that the four excretory gland peptide-like protein (EGP) encoding genes in upregulated group are structurally different than the two in the downregulated list. The amino acid composition analysis on the proteins, which were encoded by the up- or downregulated ESTs without homologues (NH) suggested that seven of the NH proteins are biochemically similar to the novel foot proteins from other mussels. The quantitative reverse transcription PCR (QRT-PCR) proved the uniqueness of the templates in the array, and also confirmed the differentially expressed genes identified by microarray experiment. Our findings demonstrated that the zebra mussel byssus cDNA microarray is an efficient tool for the studies of differential gene expression in different byssogenesis states, thereby revealing important details of the underwater adhesion.

Functional annotation of 19,841 Populus nigra full-length enriched cDNA clones.

BACKGROUND: Populus is one of favorable model plants because of its small genome. Structural genomics of Populus has reached a breakpoint as nucleotides of the entire genome have been determined. Reaching the post genome era, functional genomics of Populus is getting more important for well-comprehended plant science. Development of bioresorce serving functional genomics is making rapid progress. Huge efforts have achieved deposits of expressed sequence tags (ESTs) in various plant species consequently accelerating functional analysis of genes. ESTs from full-length cDNA clones are especially powerful for accurate molecular annotation. We promoted collection and annotation of the ESTs from Populus full-length enriched cDNA clones as part of functional genomics of tree species. RESULTS: We have been collecting the full-length enriched cDNA of the female poplar (Populus nigra var. italica) for years. By sequencing P. nigra full-length (PnFL) cDNA libraries, we generated about 116,000 5'-end or 3'-end ESTs corresponding to 19,841 nonredundant PnFL clones. Population of PnFL cDNA clones represents 44% of the predicted genes in the Populus genome. CONCLUSION: Our resource of P. nigra full-length enriched clones is expected to provide valuable tools to gain further insight into genome annotation and functional genomics in Populus.

Molecular approach to annelid regeneration: cDNA subtraction cloning reveals various novel genes that are upregulated during the large-scale regeneration of the oligochaete, Enchytraeus japonensis.

To identify genes specifically activated during annelid regeneration, suppression subtractive hybridization was performed with cDNAs from regenerating and intact Enchytraeus japonensis, a terrestrial oligochaete that can regenerate a complete organism from small body fragments within 4-5 days. Filter array screening subsequently revealed that about 38% of the forward-subtracted cDNA clones contained genes that were upregulated during regeneration. Two hundred seventy-nine of these clones were sequenced and found to contain 165 different sequences (79 known and 86 unknown). Nine clones were fully sequenced and four of these sequences were matched to known genes for glutamine synthetase, glucosidase 1, retinal protein 4, and phosphoribosylaminoimidazole carboxylase, respectively. The remaining five clones encoded an unknown open-reading frame. The expression levels of these genes were highest during blastema formation. Our present results, therefore, demonstrate the great potential of annelids as a new experimental subject for the exploration of unknown genes that play critical roles in animal regeneration.

Application of SSH and a macroarray to investigate altered gene expression in Mytilus edulis in response to exposure to benzo[a]pyrene.

The lack of genomic resources for aquatic invertebrates restricts their use as sentinel species in coastal environments. It is known that where genomic data are not available, suppression subtractive hybridisation (SSH) can generate cDNA libraries representative of pollutant-responsive gene transcription in aquatic vertebrates. To assess whether the approach was equally suited to aquatic invertebrates, altered gene expression in digestive gland of the mussel, Mytilus edulis, in response to exposure to benzo[a]pyrene (BaP) (1 mg/l) was investigated with SSH and a nylon macroarray. Screening of the subtracted libraries showed 112/250 up-regulated and 25/55 down-regulated clones were positive for differential expression and characterisation of these identified 87 with unique sequence suitable for array on a nylon membrane. The transcripts isolated were from a diverse range of genes involved in general stress, oxidative stress, cell adhesion, transcriptional and translational regulation, transport mechanisms, energy metabolism, cell metabolism, lipid metabolism, protein turnover and activation, lysosomal activity and 22 cryptic clones. Subsequent use of the clones in macroarray format to analyse expression of BaP-responsive genes (0 vs 4 day exposed) showed 0-100-fold increased levels of the forward-subtracted probes and between 0 and 0.1-fold down-regulation of the reverse-subtracted probes. Only 15% of the clones showed less than 2-fold change in expression. The gene ontology of the transcripts isolated demonstrates that BaP elicits a multitude of responses with a major feature being disruption of cellular redox status. The results indicate that the use of SSH and a macroarray is a robust method to discover novel pollutant-responsive genes in aquatic invertebrates.

Evaluation of normalization methods for cDNA microarray data by k-NN classification.

BACKGROUND: Non-biological factors give rise to unwanted variations in cDNA microarray data. There are many normalization methods designed to remove such variations. However, to date there have been few published systematic evaluations of these techniques for removing variations arising from dye biases in the context of downstream, higher-order analytical tasks such as classification. RESULTS: Ten location normalization methods that adjust spatial- and/or intensity-dependent dye biases, and three scale methods that adjust scale differences were applied, individually and in combination, to five distinct, published, cancer biology-related cDNA microarray data sets. Leave-one-out cross-validation (LOOCV) classification error was employed as the quantitative end-point for assessing the effectiveness of a normalization method. In particular, a known classifier, k-nearest neighbor (k-NN), was estimated from data normalized using a given technique, and the LOOCV error rate of the ensuing model was computed. We found that k-NN classifiers are sensitive to dye biases in the data. Using NONRM and GMEDIAN as baseline methods, our results show that single-bias-removal techniques which remove either spatial-dependent dye bias (referred later as spatial effect) or intensity-dependent dye bias (referred later as intensity effect) moderately reduce LOOCV classification errors; whereas double-bias-removal techniques which remove both spatial- and intensity effect reduce LOOCV classification errors even further. Of the 41 different strategies examined, three two-step processes, IGLOESS-SLFILTERW7, ISTSPLINE-SLLOESS and IGLOESS-SLLOESS, all of which removed intensity effect globally and spatial effect locally, appear to reduce LOOCV classification errors most consistently and effectively across all data sets. We also found that the investigated scale normalization methods do not reduce LOOCV classification error. CONCLUSION: Using LOOCV error of k-NNs as the evaluation criterion, three double-bias-removal normalization strategies, IGLOESS-SLFILTERW7, ISTSPLINE-SLLOESS and IGLOESS-SLLOESS, outperform other strategies for removing spatial effect, intensity effect and scale differences from cDNA microarray data. The apparent sensitivity of k-NN LOOCV classification error to dye biases suggests that this criterion provides an informative measure for evaluating normalization methods. All the computational tools used in this study were implemented using the R language for statistical computing and graphics.

An insight into the salivary transcriptome and proteome of the adult female mosquito Culex pipiens quinquefasciatus.

To obtain an insight into the salivary transcriptome and proteome (sialome) of the adult female mosquito Culex quinquefasciatus, a cDNA library was randomly sequenced, and aminoterminal information for selected proteins and peptides was obtained. cDNA sequence clusters coding for secreted proteins were further analyzed. The transcriptome revealed messages coding for several proteins of known families previously reported in the salivary glands of other blood-feeding insects as well as immune-related products such as C-type lectin, gambicin, and members of the prophenol oxidase cascade. Additionally, several transcripts coding for low-complexity proteins were found, some clearly coding for mucins. Many novel transcripts were found, including a novel endonuclease previously described in crabs and shrimps but not in insects; a hyaluronidase, not described before in mosquito salivary glands but found in venom glands and in salivary glands of sand flies and black flies; several cysteine-rich peptides with possible anticlotting function, including one similar to a previously described nematode family of anti-proteases; and a completely novel family of cysteine- and tryptophane-rich proteins (CWRC family) for which 12 full-length sequences are described. Also described are 14 additional novel proteins and peptides whose function and/or family affiliation are unknown. In total, 54 transcripts coding for full-length proteins are described. That several of these are translated into proteins was confirmed by finding the corresponding aminoterminal sequences in the SDS-PAGE/Edman degradation experiments. Electronic versions of all tables and sequences can be found at http://www.ncbi.nlm.nih.gov/projects/Mosquito/C_quinquefasciatus_sialome.

Negative subtraction hybridization: an efficient method to isolate large numbers of condition-specific cDNAs.

BACKGROUND: The construction of cDNA libraries is a useful tool to understand gene expression in organisms under different conditions, but random sequencing of unbiased cDNA collections is laborious and can give rise to redundant EST collections. We aimed to isolate cDNAs of messages induced by switching Aspergillus nidulans from growth on glucose to growth on selected polysaccharides. Approximately 4,700 contigs from 12,320 ESTs were already available from a cDNA library representing transcripts isolated from glucose-grown A. nidulans during asexual development. Our goals were to expand the cDNA collection without repeated sequencing of previously identified ESTs and to find as many transcripts as possible that are specifically induced in complex polysaccharide metabolism. RESULTS: We have devised a Negative Subtraction Hybridization (NSH) method and tested it in A. nidulans. NSH entails screening a plasmid library made from cDNAs prepared from cells grown under a selected physiological condition with labeled cDNA probes prepared from another physiological condition. Plasmids with inserts that failed to hybridize to cDNA probes through two rounds of screening (i.e. negatives) indicate that they are transcripts present at low concentration in the labeled probe pool. Thus, these transcripts will be predominantly condition-specific, along with some rare transcripts. In a screen for transcripts induced by switching the carbon source from glucose to 12 selected polysaccharides, 3,532 negatives were isolated from approximately 100,000 surveyed colonies using this method. Negative clones were end-sequenced and assembled into 2,039 contigs, of which 1,722 were not present in the previously characterized glucose-grown cDNA library. Single-channel microarray hybridization experiments confirmed that the majority of the negatives represented genes that were differentially induced by a switch from growth in glucose to one or more of the polysaccharides. CONCLUSIONS: The Negative Subtraction Hybridization method described here has several practical benefits. This method can be used to screen any existing cDNA library, including full-length and pooled libraries, and does not rely on PCR or sequence information. In addition, NSH is a cost-effective method for the isolation of novel, full-length cDNAs for differentially expressed transcripts or enrichment of rare transcripts.

Anomalies in the expression profile of interspecific hybrids of Drosophila melanogaster and Drosophila simulans.

When females of Drosophila melanogaster and males of Drosophila simulans are mated, the male progeny are inviable, whereas the female progeny display manifold malformations and are sterile. These abnormalities result from genetic incompatibilities accumulated since the time the lineages of the species diverged, and may have their origin in aberrant gene transcription. Because compensatory changes within species may obscure differences at the regulatory level in conventional comparisons of the expression profile between species, we have compared the gene-expression profile of hybrid females with those of females of the parental species in order to identify regulatory incompatibilities. In the hybrid females, we find abnormal levels of messenger RNA for a large fraction of the Drosophila transcriptome. These include a gross underexpression of genes preferentially expressed in females, accompanying gonadal atrophy. The hybrid females also show significant overexpression of male-biased genes, which we attribute to incompatibilities in the regulatory mechanisms that normally act to control the expression of these genes in females. The net result of the multiple incompatibilities is that the gene-expression profiles of the parental females are more similar to each other than either is to that of the hybrid.

Numerous novel annotations of the human genome sequence supported by a 5'-end-enriched cDNA collection.

A collection of 90,000 human cDNA clones generated to increase the fraction of "full-length" cDNAs available was analyzed by sequence alignment on the human genome assembly. Five hundred fifty-two gene models not found in LocusLink, with coding regions of at least 300 bp, were defined by using this collection. Exon composition proposed for novel genes showed an average of 4.7 exons per gene. In 20% of the cases, at least half of the exons predicted for new genes coincided with evolutionary conserved regions defined by sequence comparisons with the pufferfish Tetraodon nigroviridis. Among this subset, CpG islands were observed at the 5' end of 75%. In-frame stop codons upstream of the initiator ATG were present in 49% of the new genes, and 16% contained a coding region comprising at least 50% of the cDNA sequence. This cDNA resource also provided candidate small protein-coding genes, usually not included in genome annotations. In addition, analysis of a sample from this cDNA collection indicates that approximately 380 gene models described in LocusLink could be extended at their 5' end by at least one new exon. Finally, this cDNA resource provided an experimental support for annotations based exclusively on predictions, thus representing a resource substantially improving the human genome annotation.

The transcriptome of adult female Anopheles darlingi salivary glands.

Anopheles (Nyssorhynchus) darlingi is an important malaria vector in South and Central America; however, little is known about molecular aspects of its biology. Genomic and proteomic analyses were performed on the salivary gland products of Anopheles darlingi. A total of 593 randomly selected, salivary gland-derived cDNAs were sequenced and assembled based on their similarities into 288 clusters. The putative translated proteins were classified into three categories: (S) secretory products, (H) housekeeping products and (U) products with unknown cell location and function. Ninety-three clusters encode putative secreted proteins and several of them, such as an anophelin, a thrombin inhibitor, apyrases and several new members of the D7 protein family, were identified as molecules involved in haematophagy. Sugar-feeding related enzymes (alpha-glucosidases and alpha-amylase) also were found among the secreted salivary products. Ninety-nine clusters encode housekeeping proteins associated with energy metabolism, protein synthesis, signal transduction and other cellular functions. Ninety-seven clusters encode proteins with no similarity with known proteins. Comparison of the sequence divergence of the S and H categories of proteins of An. darlingi and An. gambiae revealed that the salivary proteins are less conserved than the housekeeping proteins, and therefore are changing at a faster evolutionary rate. Tabular and supplementary material containing the cDNA sequences and annotations are available at http://www.ncbi.nlm.nih.gov/projects/Mosquito/A_darlingi_sialome/