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PubMed2001

The sequence of the human genome.

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

Algorithms↗

PubMed2005

PhyloGibbs: a Gibbs sampling motif finder that incorporates phylogeny.

A central problem in the bioinformatics of gene regulation is to find the binding sites for regulatory proteins. One of the most promising approaches toward identifying these short and fuzzy sequence patterns is the comparative analysis of orthologous intergenic regions of related species. This analysis is complicated by various factors. First, one needs to take the phylogenetic relationship between the species into account in order to distinguish conservation that is due to the occurrence of functional sites from spurious conservation that is due to evolutionary proximity. Second, one has to deal with the complexities of multiple alignments of orthologous intergenic regions, and one has to consider the possibility that functional sites may occur outside of conserved segments. Here we present a new motif sampling algorithm, PhyloGibbs, that runs on arbitrary collections of multiple local sequence alignments of orthologous sequences. The algorithm searches over all ways in which an arbitrary number of binding sites for an arbitrary number of transcription factors (TFs) can be assigned to the multiple sequence alignments. These binding site configurations are scored by a Bayesian probabilistic model that treats aligned sequences by a model for the evolution of binding sites and "background" intergenic DNA. This model takes the phylogenetic relationship between the species in the alignment explicitly into account. The algorithm uses simulated annealing and Monte Carlo Markov-chain sampling to rigorously assign posterior probabilities to all the binding sites that it reports. In tests on synthetic data and real data from five Saccharomyces species our algorithm performs significantly better than four other motif-finding algorithms, including algorithms that also take phylogeny into account. Our results also show that, in contrast to the other algorithms, PhyloGibbs can make realistic estimates of the reliability of its predictions. Our tests suggest that, running on the five-species multiple alignment of a single gene's upstream region, PhyloGibbs on average recovers over 50% of all binding sites in S. cerevisiae at a specificity of about 50%, and 33% of all binding sites at a specificity of about 85%. We also tested PhyloGibbs on collections of multiple alignments of intergenic regions that were recently annotated, based on ChIP-on-chip data, to contain binding sites for the same TF. We compared PhyloGibbs's results with the previous analysis of these data using six other motif-finding algorithms. For 16 of 21 TFs for which all other motif-finding methods failed to find a significant motif, PhyloGibbs did recover a motif that matches the literature consensus. In 11 cases where there was disagreement in the results we compiled lists of known target genes from the literature, and found that running PhyloGibbs on their regulatory regions yielded a binding motif matching the literature consensus in all but one of the cases. Interestingly, these literature gene lists had little overlap with the targets annotated based on the ChIP-on-chip data. The PhyloGibbs code can be downloaded from http://www.biozentrum.unibas.ch/~nimwegen/cgi-bin/phylogibbs.cgi or http://www.imsc.res.in/~rsidd/phylogibbs. The full set of predicted sites from our tests on yeast are available at http://www.swissregulon.unibas.ch.

Algorithms↗

PubMed2002

Identification of Bartonella bacilliformis genotypes and their relevance to epidemiological investigations of human bartonellosis.

Genotypic diversity among 26 isolates of Bartonella bacilliformis obtained from different areas of Peru, and at different times, was assessed by comparison of DNA sequences derived from 16S-23S ribosomal DNA intergenic spacer regions (ISR) and a citrate synthase gene (gltA) fragment and by amplified fragment length polymorphism (AFLP) analysis. gltA comparison divided the isolates into two groups, whereas ISR comparison revealed six sequences. AFLP analysis using a selective primer delineated five profiles that correlated well with those obtained by sequence comparison. Combination of all three data sets divided the isolates into six genotypes. One of these genotypes was common to isolates collected from a large area in western Peru that corresponded to the region of endemicity for bartonellosis; however, isolates belonging to two other genotypes were also found within this region. Two of these genotypes were found in isolates isolated more than 35 years apart. The remaining three genotypes were each specifically associated with three outbreaks of bartonellosis that have recently occurred in areas where the disease had not previously been recognized. Demonstration of the unique nature of these isolates indicates that the outbreaks with which they were associated did not result from the introduction of disease by individuals who acquired their infection in the recognized region of endemicity. The sources of these outbreaks remain unknown. A consensus approach to bacterial typing using comparative sequence analysis of multiple genetic loci and the pan-genomic sampling of AFLP appears to offer a well-supported assessment of B. bacilliformis diversity, and the genotypic differences identified appear to have epidemiological significance.

Bacterial Proteins↗

PubMed1997

Molecular cloning and analysis of Schizosaccharomyces pombe Reb1p: sequence-specific recognition of two sites in the far upstream rDNA intergenic spacer.

The coding sequences for a Schizosaccharomyces pombe sequence-specific DNA binding protein, Reb1p, have been cloned. The predicted S. pombe Reb1p is 24-29% identical to mouse TTF-1 (transcription termination factor-1) and Saccharomyces cerevisiae REB1 protein, both of which direct termination of RNA polymerase I catalyzed transcripts. The S.pombe Reb1 cDNA encodes a predicted polypeptide of 504 amino acids with a predicted molecular weight of 58.4 kDa. The S. pombe Reb1p is unusual in that the bipartite DNA binding motif identified originally in S.cerevisiae and Klyveromyces lactis REB1 proteins is uninterrupted and thus S.pombe Reb1p may contain the smallest natural REB1 homologous DNA binding domain. Its genomic coding sequences were shown to be interrupted by two introns. A recombinant histidine-tagged Reb1 protein bearing the rDNA binding domain has two homologous, sequence-specific binding sites in the S. pomber DNA intergenic spacer, located between 289 and 480 nt downstream of the end of the approximately 25S rRNA coding sequences. Each binding site is 13-14 bp downstream of two of the three proposed in vivo termination sites. The core of this 17 bp site, AGGTAAGGGTAATGCAC, is specifically protected by Reb1p in footprinting analysis.

Amino Acid Sequence↗

PubMed2001

Molecular techniques open up new vistas for typing of coagulase-negative staphylococci.

Several methods were used to type 64 clinical isolates of coagulase-negative staphylococci (CNS) derived from hospitals in Morocco. The clinical isolates originated principally from blood cultures and wound sources. These isolates provided the opportunity to substantially compare the proficiency of developing molecular techniques with conventional phenotypic tests for use in the identification of clinical staphylococci. The following molecular methods were examined: Utility ribotyping analysis (Ribotyping); PCR analysis performed with 16S-23S ribosomal-DNA intergenic spacer (ITS-PCR); PCR-based random amplified polymorphic DNA (RAPD). The results obtained by the molecular techniques were contrasted to those of conventional phenotypic tests. Conventional phenotypic tests allowed the outright recognition of the majority of isolates (50/64). These 50 isolates were subdivided into 33 novobiocin-susceptible and 17 novobiocin-resistant strains of CNS. However, 2 other novobiocin-susceptible and 12 other novobiocin-resistant isolates remained unclassified by these tests. There was a good agreement between the conventional phenotypic tests and RAPD for the 33 novobiocin-susceptible isolates. But, the RAPD technique permitted the assignment of the two unidentified novobiocin-susceptible isolates to the Staphylococcus hominis species. A complete correlation was obtained between the three molecular tools for recognition of the 12 novobiocin-resistant isolates that were not identified by phenotypic typing; these were in fact identified as 5 Staphylococcus cohnii and 4 Staphylococcus equorum. Three isolates remained unidentified by all three systems of molecular techniques.

Bacterial Proteins↗

PubMed1996

Characterization of indole-negative Bacteroides fragilis group species with use of polymerase chain reaction fingerprinting and resistance profiles.

Biochemical tests alone do not adequately differentiate the various Bacteroides species, groups, and antimicrobial-resistant variants. Consequently, we used a polymerase chain reaction (PCR) fingerprinting technique, with either a single nonspecific primer derived from the t-DNA intergenic spacer region (T3B) or a single primer that anneals to minisatellite DNA sequences (M13 core), to identify and characterize 58 clinical isolates of Bacteroides fragilis group species (B. fragilis, B. distasonis, and B. caccae). In addition to species- and subspecies-specific differences, 4 strains of B. fragilis, 1 of B. distasonis, and 3 of B. caccae that showed increased resistance to imipenem, ampicillin, and ampicillin/sulbactam also produced unique PCR fingerprint profiles. Analysis by the clinical source of isolation (i.e. blood or intraabdominal, skin, or soft-tissue infection) indicated that no particular PCR fingerprint type was associated with greater pathogenicity of any individual clinical source. The PCR fingerprinting technique proves to be a useful tool for species identification and taxonomic studies, as well as for epidemiological studies of Bacteroides species.

Bacterial Typing Techniques↗

PubMed2006

Phylogenetic analysis of the rDNA intergenic spacer subrepeats and its implication for the domestication history of foxtail millet, Setaria italica.

We sequenced ribosomal DNA intergenic spacer subrepeats and their flanking regions of foxtail millet landraces from various regions in Europe and Asia and its wild ancestor to elucidate phylogenetic differentiation within each of types I-III found in our previous work and to elucidate relationships among these three types. Type I was classified into seven subtypes designated as Ia-Ig based on subrepeat sequences; C repeats downstream of those subrepeats are also polymorphic. Of these, subtypes Ia-Id and Ig were found in foxtail millet landraces. Subtypes Ia and Ib were distributed broadly throughout Asia and Europe. Subtype Ic was distributed in China, Korea and Japan. Subtype Id has a 20-bp deletion in subrepeat 3 and has a unique C repeat sequence. This subtype was found in a morphologically primitive landrace group from Afghanistan and northwestern Pakistan and differed greatly from other type I subtypes, implying that these landraces were domesticated independently. Subtypes Ig was found in a landrace from Pakistan and Ia and Ie-Ig were in six wild ancestor accessions. Type II was also highly polymorphic and four subtypes were found and designated as subtypes IIa-IId, but sequence analyses indicated type III as monomorphic. The present work indicates that type III should be classified as a subtype of type II (subtype IIe). Sequence polymorphism of subrepeats of types I-III indicated that subrepeats of subtype IIa are greatly divergent from others. Relationships among types I-III are much more complicated than anticipated based on previous RFLP work.

Base Sequence↗

PubMed1998

Evidence of multiple transcription initiation and termination sites within the rDNA intergenic spacer and rRNA readthrough transcription in the urochordate Herdmania curvata.

Analysis of the structure of the urochordate Herdmania curvata ribosomal DNA intergenic spacer (IGS) and its role in transcription initiation and termination suggests that rRNA gene regulation in this chordate differs from that in vertebrates. A cloned H. curvata IGS is 1881 bp and composed predominantly of two classes of similar repeat sequences that largely alternate in a tandem array. Southern blot hybridization demonstrates that the IGS length variation within an individual and population is largely the result of changes in internal repeat number. Nuclease S1 mapping and primer extension analyses suggest that there are two transcription initiation sites at the 3' end of the most 3' repetitive element; these sites are 6 nucleotides apart. Unlike mouse, Xenopus, and Drosophila, there is no evidence of transcription starting elsewhere in the IGS. Most sequence differences between the promoter repeat and the other internal repeats are in the vicinity of the putative initiation sites. As in Drosophila, nuclease S1 mapping of transcription termination sites suggest that there is not a definitive stop site and a majority of the pre-rRNAs read through a substantial portion of the IGS. Some transcription appears to proceed completely through the promoter repeat into the adjacent rDNA unit. Analysis of oocyte RNA by reverse transcription-polymerase chain reaction (RT-PCR) confirms that readthrough transcription into the adjacent rDNA unit is occurring in some small IGS length variants; there is no evidence of complete readthrough of IGSs larger than 1.0 kb.

Animals↗

PubMed2002

Phylogenetics of Miscanthus, Saccharum and related genera (Saccharinae, Andropogoneae, Poaceae) based on DNA sequences from ITS nuclear ribosomal DNA and plastid trnLintron and trnL-F intergenic spacers.

DNA sequences were used to assess the monophyly and inter-relationships of Miscanthus, Saccharumand related genera in the Saccharum complex. Three DNA regions were sequenced, including the trnLintron and the trnL-F intergenic spacer of the plastid genome and the ITS region of nuclear ribosomal DNA (nrDNA). Because it was more variable, the ITS region proved most suitable for phylogenetic reconstruction at this level, and the results indicate that Miscanthus s.l. and Saccharum s.l. are polyphyletic. A set of species from Saccharum section Ripidium(clade a) do not group closely with any members of Saccharum s.l. A number of Miscanthus species from eastern or south-eastern Asia represent a monophyletic group with a basic chromosome number of 19 (clade b), but the other species from Africa and the Himalayas are clearly excluded. There is support for a monophyletic Saccharum s.s. clade including S. officinarumand S. spontaneum that is sister to Miscanthus s.s(clade c). There is no evidence to support the division of some Saccharum s.l. into the genera currently known as Erianthus and Narenga. Saccharum contortum( =Erianthus contortus), S. narenga (= Narenga porphyrocoma) and Erianthus rockii, group more closely with Miscanthus fuscus, a species from the Himalayas and also with the African Miscanthus s.l. species (= Miscanthidium, clade d).

Journal Article↗

PubMed2004

Development of a species-specific probe for Pythium insidiosum and the diagnosis of pythiosis.

Pythium insidiosum, the only species in the genus that infects mammals, is the etiological agent of pythiosis, a granulomatous disease characterized by cutaneous and subcutaneous lesions and vascular diseases. Accurate diagnosis of pythiosis and identification of its causal agent are often inconsistent with current immunological diagnostic methods. A species-specific DNA probe was constructed by using a 530-bp HinfI fragment from the ribosomal DNA intergenic spacer of P. insidiosum. When the probe was incubated with dot blots of genomic DNA from 104 Pythium species, it hybridized only to the DNA of P. insidiosum and P. destruens-two species that have been considered conspecific. The probe also hybridized to DNA from 22 P. insidiosum isolates in this study, regardless of their geographic origin or animal host. When tested against genomic DNA from other pathogenic organisms (Aspergillus fumigatus, Basidiobolus ranarum, Conidiobolus coronatus, Lagenidium giganteum, Paracoccidioides brasiliensis, and Prototheca wickerhamii), no cross-hybridization of the probe was detected. The specificity of the probe to hybridize to genomic DNA from all isolates of P. insidiosum and not cross-react with DNA from other Pythium species or pathogens that cause symptoms similar to pythiosis in their hosts makes it a powerful tool for the accurate diagnosis of pythiosis. In addition, the probe has the potential for pathological and environmental diagnostic applications.

DNA Probes↗

PubMed2003

Genetic diversity among clinical isolates of Acremonium strictum determined during an investigation of a fatal mycosis.

Primarily saprophytic in nature, fungi of the genus Acremonium are a well-documented cause of mycetoma and other focal diseases. More recently, a number of Acremonium spp. have been implicated in invasive infections in the setting of severe immunosuppression. During the course of routine microbiological studies involving a case of fatal mycosis in a nonmyeloablative hematopoietic stem cell transplant patient, we identified a greater-than-expected variation among strains previously identified as Acremonium strictum by clinical microbiologists. Using DNA sequence analysis of the ribosomal DNA intergenic transcribed spacer (ITS) regions and the D1-D2 variable domain of the 28S ribosomal DNA gene (28S), the case isolate and four other clinical isolates phenotypically identified as A. strictum were found to have <99% homology to the A. strictum type strain, CBS 346.70, at the ITS and 28S loci, while a sixth isolate phenotypically identified only as Acremonium sp. had >99% homology to the type strain at both loci. These results suggest that five out of the six clinical isolates belong to species other than A. strictum or that the A. strictum taxon is genetically diverse. Based upon these sequence data, the clinical isolates were placed into three genogroups.

Acremonium↗

PubMed1987

Sequence dependence of Drosophila topoisomerase II in plasmid relaxation and DNA binding.

The sequence dependence of Drosophila topoisomerase II supercoil relaxation and binding activities has been examined. The DNA substrates used in binding experiments were two fragments from Drosophila heat shock locus 87A7. One of these DNA fragments includes the coding region for the heat shock protein hsp70, and the other includes the intergenic non-coding region that separates two divergently transcribed copies of the hsp70 gene at the locus. The intergenic region was previously shown to have a much higher density of topoisomerase cleavage sites than the hsp70 coding region. Competition nitrocellulose filter binding assays demonstrate a preferential binding of the intergene fragment, and that binding specificity increases with increasing ionic strength. Dissociation kinetics indicate a greater kinetic stability of topoisomerase II complexes with the intergene DNA fragment. To study topoisomerase II relaxation activity, we used supercoiled plasmids that contained the same fragments from locus 87A7 cloned as inserts. The relative relaxation rates of the two plasmids were determined under several conditions of ionic strength, and when the plasmid substrates were included in separate reactions or when they were mixed in a single reaction. The relaxation properties of these two plasmids can be explained by a coincidence of high-affinity binding sites, strong cleavage sites, and sites used during the catalysis of strand passage events by topoisomerase II. Sequence dependence of topoisomerase II catalytic activity may therefore parallel the sequence dependence of DNA cleavage by this enzyme.

Animals↗

PubMed2000

Computational and experimental characterization of physically clustered simple sequence repeats in plants.

The type and frequency of simple sequence repeats (SSRs) in plant genomes was investigated using the expanding quantity of DNA sequence data deposited in public databases. In Arabidopsis, 306 genomic DNA sequences longer than 10 kb and 36,199 EST sequences were searched for all possible mono- to pentanucleotide repeats. The average frequency of SSRs was one every 6.04 kb in genomic DNA, decreasing to one every 14 kb in ESTs. SSR frequency and type differed between coding, intronic, and intergenic DNA. Similar frequencies were found in other plant species. On the basis of these findings, an approach is proposed and demonstrated for the targeted isolation of single or multiple, physically clustered SSRs linked to any gene that has been mapped using low-copy DNA-based markers. The approach involves sample sequencing a small number of subclones of selected randomly sheared large insert DNA clones (e.g., BACs). It is shown to be both feasible and practicable, given the probability of fortuitously sequencing through an SSR. The approach is demonstrated in barley where sample sequencing 34 subclones of a single BAC selected by hybridization to the Big1 gene revealed three SSRs. These allowed Big1 to be located at the top of barley linkage group 6HS.

Arabidopsis↗

PubMed1997

Low-resolution sequencing of Rhodobacter sphaeroides 2.4.1T: chromosome II is a true chromosome.

The photosynthetic bacterium Rhodobacter sphaeroides 2.4.1T has two chromosomes, CI (approximately 3.0 Mb) and CII (approximately 0.9 Mb). In this study a low-redundancy sequencing strategy was adopted to analyse 23 out of 47 cosmids from an ordered CII library. The sum of the lengths of these 23 cosmid inserts was approximately 495 kb, which comprised approximately 417 kb of unique DNA. A total of 1145 sequencing runs was carried out, with each run generating 559 +/- 268 bases of sequence to give approximately 640 kb of total sequence. After editing, approximately 2.8% bases per run were estimated to be ambiguous. After the removal of vector and Escherichia coli sequences, the remaining approximately 565 kb of R. sphaeroides sequences were assembled, generating approximately 291 kb of unique sequences. BLASTX analysis of these unique sequences suggested that approximately 131 kb (45% of the unique sequence) had matches to either known genes, or database ORFs of hypothetical or unknown function (dORFs). A total of 144 strong matches to the database was found; 101 of these matches represented genes encoding a wide variety of functions, e.g. amino acid biosynthesis, photosynthesis, nutrient transport, and various regulatory functions. Two rRNA operons (rrnB and rrnC) and five tRNAs were also identified. The remaining 160 kb of DNA sequence which did not yield database matches was then analysed using CODONPREFERENCE from the GCG package. This analysis suggested that 122 kb (42% of the total unique DNA sequence) could encode putative ORFs (pORFs), with the remaining 38 kb (13%) possibly representing non-coding intergenic DNA. From the data so far obtained, CII does not appear to be specialized for encoding any particular metabolic function, physiological state or growth condition. These data suggest that CII contains genes which are functionally as diverse as those found on any other bacterial chromosome and also contains sequences (pORFs), which may prove to be unique to this organism.

Base Sequence↗

PubMed1989

Nucleotide sequence of the rat gamma-crystallin gene region and comparison with an orthologous human region.

The sequences of a 51-kb region containing the cluster of five rat gamma-crystallin-coding genes (CRYG) and of a 7-kb region surrounding the sixth rat CRYG gene were determined. Approximately 78% of the total sequence represents intergenic DNA. We also sequenced 22 kb of DNA from the human CRYG gene cluster. All CRYG genes are associated with CpG-rich regions. The sequence similarity between the human and rat gene regions drops sharply (to 65%) in intronic and 3'-flanking regions but decreases only gradually in the 5'-flanking region. Highly conserved regions (greater than 80%) are found as far upstream as 1.5 kb. Overall intergenic distances are conserved. The human region contains much more repetitive DNA (24% vs. 10%) but less simple-sequence (sps) DNA (0.7% vs. 4%) than the rat region. Almost all repeats and spsDNA elements are located in the intergenic region. The location of repetitive and spsDNA differs between the orthologous regions and these elements were probably inserted after the evolutionary separation of rat and man. The Alu repeats in man and the B3 repeats in the rat are close copies of their respective consensus sequences and bordered by virtually perfect repeats. In contrast, the B1 and B2 repeats in the rat have diverged considerably from the consensus sequence and the surrounding direct repeats are usually imperfect. Thus the dispersion of the B1 and B2 repeats in the rat probably preceded that of the B3 repeats. Within the rat genomic region the spacing of Z-DNA elements is surprisingly regular, they are located about 12 kb apart. A search for putative matrix-associated regions suggests that the rat CRYG gene cluster is organized into two chromosomal domains.

Animals↗

PubMed2001

egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus.

The recently described staphylococcal enterotoxins (SE) G and I were originally identified in two separate strains of Staphylococcus aureus. We have previously shown that the corresponding genes seg and sei are present in S. aureus in tandem orientation, on a 3.2-kb DNA fragment (Jarraud, J. et al. 1999. J. Clin. Microbiol. 37:2446-2449). Sequence analysis of seg-sei intergenic DNA and flanking regions revealed three enterotoxin-like open reading frames related to seg and sei, designated sek, sel, and sem, and two pseudogenes, psi ent1 and psi ent2. RT-PCR analysis showed that all these genes, including seg and sei, belong to an operon, designated the enterotoxin gene cluster (egc). Recombinant SEG, SEI, SEK, SEL, and SEM showed superantigen activity, each with a specific V beta pattern. Distribution studies of genes encoding superantigens in clinical S. aureus isolates showed that most strains harbored such genes and in particular the enterotoxin gene cluster, whatever the disease they caused. Phylogenetic analysis of enterotoxin genes indicated that they all potentially derived from this cluster, identifying egc as a putative nursery of enterotoxin genes.

3' Untranslated Regions↗

PubMed1993

Identification of single specimens of the Anopheles gambiae complex by the polymerase chain reaction.

A ribosomal DNA-polymerase chain reaction (PCR) method has been developed for species identification of individuals of the five most widespread members of the Anopheles gambiae complex, a group of morphologically indistinguishable sibling mosquito species that includes the major vectors of malaria in Africa. The method, which is based on species-specific nucleotide sequences in the ribosomal DNA intergenic spacers, may be used to identify both species and interspecies hybrids, regardless of life stage, using either extracted DNA or fragments of a specimen. Intact portions of a mosquito as small as an egg or the segment of one leg may be placed directly into the PCR mixture for amplification and analysis. The method uses a cocktail of five 20-base oligonucleotides to identify An. gambiae, An. arabiensis, An. quadriannnulatus, and either An. melas in western Africa or An. melas in eastern and southern Africa.

Animals↗

PubMedDate not supplied

Structure and organization of the human globin genes.

A very precise picture of the fine structure and organization of the human globin genes has recently emerged from the study of cloned fragments of chromosomal DNA containing the globin genes and their flanking DNA. The precise sizes, locations and nucleotide sequences of intervening sequences (introns) that interrupt the globin genes have been determined. A number of presumably functionally important preserved nucleotide sequences common to all of the globin genes have been identified within the 5'-flanking DNA of the genes, as well as in their untranslated sequences and at the junctions between coding and intervening sequences. Intergene distances as well as the sizes of duplication units of closely related genes have been determined. A somewhat unexpected finding has been the identification of apparently nonexpressed globin pseudogenes within the globin gene clusters. Finally, detailed analysis of intergene DNA has revealed the presence of different types of repetitive DNA sequences in the vicinity of many of the globin genes but the significance of this finding with regard to the control of globin gene expression is not yet known.

Amino Acid Sequence↗