We report on the development of a PCR-based assay to detect Pasteuria penetrans infection of Meloidogyne arenaria in planta using specific primers for recently sequenced sigE, spoIIAB and atpF genes of P. penetrans biotype P20. Amplification of these genes in crude DNA extracts of ground tomato root galls using real-time kinetic PCR distinguished infected from uninfected M. arenaria race 1 by analysis of consensus thresholds for single copy genes. Fluorescent in situ hybridization (FISH) using the sigE primer sequence as a probe shows hybridization to P. penetrans cells in various stages of vegetative (pre-endospore) development. Ratios of gene copies for sigE and 16S rDNA were obtained for P. penetrans and compared to Bacillus subtilis as a genomic paradigm of endospore-forming bacteria. Phylogenetic analysis of the sigE gene from Gram-positive, endospore-forming bacteria finds P. penetrans most closely related Paenbacillus polymyxa. The sporulation genes (spo genes), particularly sigE, have sequence diversity that recommends them for species and biotype differentiation of the numerous Pasteuria isolates that infect a large number of plant-parasitic nematodes.

The GenBank (Registered Trademark symbol) sequence database incorporates DNA sequences from all available public sources, primarily through the direct submission of sequence data from individual laboratories and from large-scale sequencing projects. Most submitters use the BankIt (Web) or Sequin programs to format and send sequence data. Data exchange with the EMBL Data Library and the DNA Data Bank of Japan helps ensure comprehensive worldwide coverage. GenBank data is accessible through NCBI's integrated retrieval system, Entrez, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome and protein structure information. MEDLINE (Registered Trademark symbol) s from published articles describing the sequences are included as an additional source of biological annotation through the PubMed search system. Sequence similarity searching is offered through the BLAST series of database search programs. In addition to FTP, Email, and server/client versions of Entrez and BLAST, NCBI offers a wide range of World Wide Web retrieval and analysis services based on GenBank data. The GenBank database and related resources are freely accessible via the URL: http://www.ncbi.nlm.nih.gov

In most PCR-based tissue typing techniques the PCR amplification is followed by a post-amplification specificity step. In typing by PCR amplification with sequence-specific primers (PCR-SSP), typing specificity is part of the amplification step, which makes the technique almost as fast as serological tissue typing. In the present study primers were designed for DR "low-resolution" typing by PCR-SSP, i.e. identifying polymorphism corresponding to the serologically defined series DR1-DRw18. This resolution was achieved by performing 19 PCR reactions per individual, 17 for assigning DR1-DRw18 and 2 for the DRw52 and DRw53 superspecificities. Thirty cell lines and 121 individuals were typed by the DR "low-resolution" PCR-SSP technique, TaqI DRB-DQA-DQB RFLP analysis and serology. The concordance between PCR-SSP typing and RFLP analysis was 100%. The reproducibility was 100% in 40 samples typed on two separate occasions. No false-positive or false-negative typing results were obtained. All homozygous and heterozygous combinations of DR1-DRw18 could be distinguished. Amplification patterns segregated according to dominant Mendelian inheritance. DNA preparation, PCR amplification and post-amplification processing, including gel detection, documentation and interpretation, were performed in 2 hours. In conclusion, PCR-SSP is an accurate typing technique with high sensitivity, specificity and reproducibility. The method is rapid and inexpensive. DR "low-resolution" typing by the PCR-SSP technique is ideally suited for analyzing small numbers of samples simultaneously and is an alternative to serological DR typing in routine clinical practice including donor-recipient matching in cadaveric transplantations.

Two viruses, GB virus A (GBV-A) and GB virus B (GBV-B), were recently identified in the GB hepatitis agent. Human sera containing antibodies that recognize GBV-A and/or GBV-B recombinant proteins were subjected to polymerase chain reaction studies with degenerate oligonucleotides capable of amplifying a segment of the putative helicase genes from GBV-A, GBV-B or hepatitis C virus. Novel sequences related to members of the Flaviviridae were identified in sera from 12 individuals including 4 individuals with hepatitis. The limited nucleotide sequence identity between GBV-A, GBV-B and HCV sequences suggests that a novel virus, tentatively named GB virus C, may be responsible for some cases of non-A, non-B, non-C, non-D, non-E hepatitis.

The p53 tumour suppressor is a transcription factor that regulates the progression of the cell through its cycle and cell death (apoptosis) in response to environmental stimuli such as DNA damage and hypoxia. Even though p53 modulates these critical cellular processes, mice that lack p53 are developmentally normal, suggesting that p53-related proteins might compensate for the functions of p53 during embryogenesis. Two p53 homologues, p63 and p73, are known and here we describe the function of p63 in vivo. Mice lacking p63 are born alive but have striking developmental defects. Their limbs are absent or truncated, defects that are caused by a failure of the apical ectodermal ridge to differentiate. The skin of p63-deficient mice does not progress past an early developmental stage: it lacks stratification and does not express differentiation markers. Structures dependent upon epidermal-mesenchymal interactions during embryonic development, such as hair follicles, teeth and mammary glands, are absent in p63-deficient mice. Thus, in contrast to p53, p63 is essential for several aspects of ectodermal differentiation during embryogenesis.

A variety of cytokines, lymphokines and growth factors function by interacting with receptors that are members of the cytokine receptor superfamily. These receptors share extracellular motifs and have limited similarity in their cytoplasmic domains. Although lacking catalytic domains, this family of receptors couples ligand binding with the induction of tyrosine phosphorylation. Recent studies have shown that this is mediated by members of the Janus kinase (JAK) family of cytoplasmic protein tyrosine kinases. JAKs physically associate with the membrane-proximal region of the ligand-bound receptor, leading to their tyrosine phosphorylation and activation. The activated JAKs phosphorylate the receptors as well as cytoplasmic proteins belonging to a family of transcription factors called the signal transducers and activators of transcription (STATs), providing a novel signaling pathway that is shared by all members of the cytokine receptor superfamily.

Mesoderm formation is critical for the establishment of the animal body plan and in Drosophila requires the snail gene. This report concerns the cloning and expression pattern of the structurally similar gene snail1 from zebrafish. In situ hybridization shows that the quantity of snail1 RNA increases at the margin of the blastoderm in cells that involute during gastrulation. As gastrulation begins, snail1 RNA disappears from the dorsal axial mesoderm and becomes restricted to the paraxial mesoderm and the tail bud. snail1 RNA increases in cells that define the posterior border of each somite and then disappears when somitic cells differentiate. Later in development, expression appears in cephalic neural crest derivatives. Many snail1-expressing cells were missing from mutant spadetail embryos and the quantity of snail1 RNA was greatly reduced in mutant no tail embryos. The work presented here suggests that snail1 is involved in morphogenetic events during gastrulation, somitogenesis and development of the cephalic neural crest, and that no tail may act as a positive regulator of snail1.

All vertebrates possess anatomical features not seen in their closest living relatives, the protochordates (tunicates and amphioxus). Some of these features depend on developmental processes or cellular behaviours that are again unique to vertebrates. We are interested in the genetic changes that may have permitted the origin of these innovations. Gene duplication, followed by functional divergence of new genes, may be one class of mutation that permits major evolutionary change. Here we examine the hypothesis that gene duplication events occurred close to the origin and early radiation of the vertebrates. Genome size comparisons are compatible with the occurrence of duplications close to vertebrate origins; more precise insight comes from cloning and phylogenetic analysis of gene families from amphioxus, tunicates and vertebrates. Comparisons of Hox gene clusters, other homeobox gene families, Wnt genes and insulin-related genes all indicate that there was a major phase of gene duplication close to vertebrate origins, after divergence from the amphioxus lineage; we suggest there was probably a second phase of duplication close to jawed vertebrate origins. From amphioxus and vertebrate homeobox gene expression patterns, we suggest that there are multiple routes by which new genes arising from gene duplication acquire new functions and permit the evolution of developmental innovations.

Of the sequence comparison methods, profile-based methods perform with greater selectively than those that use pairwise comparisons. Of the profile methods, hidden Markov models (HMMs) are apparently the best. The first part of this paper describes calculations that (i) improve the performance of HMMs and (ii) determine a good procedure for creating HMMs for sequences of proteins of known structure. For a family of related proteins, more homologues are detected using multiple models built from diverse single seed sequences than from one model built from a good alignment of those sequences. A new procedure is described for detecting and correcting those errors that arise at the model-building stage of the procedure. These two improvements greatly increase selectivity and coverage.The second part of the paper describes the construction of a library of HMMs, called SUPERFAMILY, that represent essentially all proteins of known structure. The sequences of the domains in proteins of known structure, that have identities less than 95 %, are used as seeds to build the models. Using the current data, this gives a library with 4894 models.The third part of the paper describes the use of the SUPERFAMILY model library to annotate the sequences of over 50 genomes. The models match twice as many target sequences as are matched by pairwise sequence comparison methods. For each genome, close to half of the sequences are matched in all or in part and, overall, the matches cover 35 % of eukaryotic genomes and 45 % of bacterial genomes. On average roughly 15% of genome sequences are labelled as being hypothetical yet homologous to proteins of known structure. The annotations derived from these matches are available from a public web server at: http://stash.mrc-lmb.cam.ac.uk/SUPERFAMILY. This server also enables users to match their own sequences against the SUPERFAMILY model library.

Distinct classes of primary sensory neurons in dorsal root ganglia subserve different sensory modalities, terminate in different dorsoventral locations in the spinal cord, and display different neurotrophin response profiles. Large diameter muscle afferents that terminate in the ventral spinal cord are NT-3 responsive, whereas small diameter afferents subserving pain and temperature are NGF responsive and terminate in the dorsal spinal cord. Previous in vitro studies showed that the developing ventral spinal cord secretes a diffusible factor that inhibits the growth of sensory axons. Here we show that this factor repels NGF-responsive axons but has little effect on NT-3-responsive axons. We also provide evidence implicating semaphorin III/collapsin, a diffusible guidance molecule expressed by ventral spinal cord cells, in mediating this effect. These results suggest that semaphorin III functions to pattern sensory projections by selectively repelling axons that normally terminate dorsally.

RNA interference (RNAi) mediated by short interfering RNAs (siRNAs) is a widely used method to analyze gene function. To use RNAi knockdown accurately to infer gene function, it is essential to determine the specificity of siRNA-mediated RNAi. We have assessed the specificity of 10 different siRNAs corresponding to the MEN1 gene by examining the expression of two additional genes, TP53 (p53) and CDKN1A (p21), which are considered functionally unrelated to menin but are sensitive markers of cell state. MEN1 RNA and corresponding protein levels were all reduced after siRNA transfection of HeLa cells, although the degree of inhibition mediated by individual siRNAs varied. Unexpectedly, we observed dramatic and significant changes in protein levels of p53 and p21 that were unrelated to silencing of the target gene. The modulations in p53 and p21 levels were not abolished on titration of the siRNAs, and similar results were obtained in three other cell lines; in none of the cell lines tested did we see an effect on the protein levels of actin. These data suggest that siRNAs can induce nonspecific effects on protein levels that are siRNA sequence dependent but that these effects may be difficult to detect until genes central to a pivotal cellular response, such as p53 and p21, are studied. We find no evidence that activation of the double-stranded RNA-triggered IFN-associated antiviral pathways accounts for these effects, but we speculate that partial complementary sequence matches to off-target genes may result in a micro-RNA-like inhibition of translation.