Showing papers on "Sequence analysis published in 1993"

Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence

Abstract: Summary Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of an extracellular signal molecule (autoinducer) in the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode functions necessary for the synthesis of, and response to, a signal molecule. Sequence analysis of the region encoding these functions revealed three open reading frames, two (luxL and luxM) that are required for production of an autoinducer substance and a third (luxN) that is required for response to this signal substance. The LuxL and LuxM proteins are not similar in amino acid sequence to other proteins in the database, but the LuxN protein contains regions of sequence resembling both the histidine protein kinase and the response regulator domains of the family of two–component, signal transduction proteins. The phenotypes of mutants with luxL, luxM and luxN defects indicated that an additional signal–response system controlling density-dependent expression of luminescence remains to be identified.

Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms.

Abstract: To facilitate genus and species level identification of a broad range of bacteria without the requirement of presumptive identification, we have developed a unified set of primers and polymerase chain reaction conditions to amplify spacer regions between the 16S and 23S genes in the prokaryotic rRNA genetic loci. Spacer regions within these loci show a significant level of length and sequence polymorphism across both genus and species lines. A generic pair of priming sequences was selected for the amplification of these polymorphisms from highly conserved sequences in the 16S and 23S genes occurring adjacent to these polymorphic regions. This single set of primers and reaction conditions was used for the amplification of 16S-23S spacer regions for over 300 strains of bacteria belonging to eight genera and 28 species or serotypes, including Listeria, Staphylococcus, and Salmonella species and additional species related to these pathogenic organisms. When the spacer amplification products were resolved by electrophoresis, the resulting patterns could be used to distinguish all of the species of bacteria within the test group. Unique elements in the amplification product patterns generally clustered at the species level, although some genus-specific characteristics were also observed. On the basis of the results obtained with our test group of 300 bacterial strains, amplification of the 16S-23S ribosomal spacer region is a suitable process for generating a data base for use in a polymerase chain reaction-based identification method, which can be comprehensively applied to the bacterial kingdom.

Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: evidence of linkage to cytotoxin production.

Abstract: A high-molecular-mass (120- to 128-kDa) Helicobacter pylori antigen has been associated with peptic ulcer disease. We created a bank of 40,000 random chromosomal fragments of H. pylori 84-183 by using lambda ZapII. Screening of this bank in Escherichia coli XL1-Blue with absorbed serum from an H. pylori-infected person permitted the isolation and purification of a clone with a 3.5-kb insert. Subcloning of this insert (pMC3) permitted the expression of a recombinant H. pylori protein that had a mass of approximately 96 kDa and that was recognized by the human serum. Sera that were obtained from H. pylori-infected persons and that recognized the native 120- to 128-kDa H. pylori antigen recognized the recombinant 96-kDa pMC3 protein to a significantly greater extent than did sera that did not recognize the native H. pylori antigen. All 19 H. pylori isolates producing the 120- to 128-kDa antigen hybridized with pMC3; none of 13 nonproducers did so (P < 0.001). Because all 15 isolates producing the vacuolating cytotoxin hybridized with pMC3, we called the gene cagA (cytotoxin-associated gene). Sequence analysis of pMC3 identified an open reading frame of 859 amino acids, without a termination codon. Parallel screening of a lambda gt11 library with human serum revealed positive plaques with identical 0.6-kb inserts and sequences matching the sequence of the downstream region of pMC3. To clone the full-length gene, we used the 0.6-kb fragment as a probe and isolated a clone with a 2.7-kb insert from the lambda ZapII genomic library. Nucleotide sequencing of this insert (pYB 2) revealed a 785-bp sequence that overlapped the downstream region of pMC3. Translation of the complete nucleotide sequence of cagA revealed an open reading frame of 1,181 amino acids yielding a protein of 131,517 daltons. There was no significant homology with any previously reported protein sequence. These findings indicate the cloning and characterization of a high-molecular-mass H. pylori antigen potentially associated with virulence and with cytotoxin production.

Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation

Abstract: An algorithm is described for the systematic characterization of the physico-chemical properties seen at each position in a multiple protein sequence alignment. The new algorithm allows questions important in the design of mutagenesis experiments to be quickly answered since positions in the alignment that show unusual or interesting residue substitution patterns may be rapidly identified. The strategy is based on a flexible set-based description of amino acid properties, which is used to define the conservation between any group of amino acids. Sequences in the alignment are gathered into subgroups on the basis of sequence similarity, functional, evolutionary or other criteria. All pairs of subgroups are then compared to highlight positions that confer the unique features of each subgroup. The algorithm is encoded in the computer program AMAS (Analysis of Multiply Aligned Sequences) which provides a textual summary of the analysis and an annotated (boxed, shaded and/or coloured) multiple sequence alignment. The algorithm is illustrated by application to an alignment of 67 SH2 domains where patterns of conserved hydrophobic residues that constitute the protein core are highlighted. The analysis of charge conservation across annexin domains identifies the locations at which conserved charges change sign. The algorithm simplifies the analysis of multiple sequence data by condensing the mass of information present, and thus allows the rapid identification of substitutions of structural and functional importance.

An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence.

Abstract: An Arabidopsis cDNA (Atmyb2) that contains a sequence that encodes a transcription factor, which is a homolog of MYB, was cloned from a cDNA library prepared from dehydrated Arabidopsis rosette plants. A gene (Atmyb2) corresponding to the Atmyb2 cDNA was also cloned and its nucleotide sequence was determined. RNA gel blot analysis showed that the Atmyb2 mRNA was induced by dehydration and disappeared upon rehydration. The Atmyb2 mRNA also accumulated upon salt stress and with the onset of treatment with abscisic acid. A beta-glucuronidase reporter gene driven by the Atmyb2 promoter was induced by dehydration and salt stress in transgenic Arabidopsis plants. These observations indicate that Atmyb2 is responsive to dehydration at the transcriptional level. The putative protein (ATMYB2) encoded by Atmyb2 has 274 amino acids, a molecular mass of 32 kD, and a putative DNA binding domain that shows considerable homology to plant MYB-related proteins, such as maize C1. A fusion protein that included ATMYB2 was expressed in Escherichia coli, and it bound specifically to oligonucleotides that contained a consensus MYB recognition sequence (TAACTG), such as is found in the simian virus 40 enhancer and the maize bronze-1 promoter. Binding was sequence specific, as indicated by a gel mobility shift experiment. These results suggest that a MYB-related transcription factor is involved in the regulation of genes that are responsive to water stress in Arabidopsis.

Characterization of the nisin gene cluster nisABTCIPR of Lactococcus lactis. Requirement of expression of the nisA and nisI genes for development of immunity

Abstract: The nisin gene cluster nisABTCIPR of Lactococcus lactis, located on a 10-kbp DNA fragment of the nisin-sucrose transposon Tn5276, was characterized. This fragment was previously shown to direct nisin-A biosynthesis and to contain the nisP and nisR genes, encoding a nisin leader peptidase and a positive regulator, respectively [van der Meer, J. R., Polman, J., Beerthuyzen, M. M., Siezen, R. J., Kuipers, O. P. & de Vos, W. M. (1993) J. Bacteriol. 175, 2578–2588]. Further sequence analysis revealed the presence of four open-reading frames, nisB, nisT, nisC and nisI, downstream of the structural gene nisA. The nisT, nisC and nisI genes were subcloned and expressed individually in Escherichia coli, using the T7-RNA-polymerase system. This resulted in the production of radio-labelled proteins with sizes of 45 kDa (NisC) and 32 kDa (NisI). The nisT gene product was not detected, possibly because of protein instability. The deduced amino acid sequence of NisI contained a consensus Iipoprotein signal sequence, suggesting that this protein is a lipid-modified extracellular membrane-anchored protein. Expression of nisI in L. Iactis provided the cells with a significant level of protection against exogeneously added nisin, indicating that NisI plays a role in the immunity mechanism. In EDTA-treated E. coli cells, expression of nisI conferred up to a 170-fold increase in immunity against nisin A compared to controls. Moreover, a lactococcal strain deficient in nisin-A production, designated NZ9800, was created by gene replacement of nisA by a truncated nisA gene and was 10-fold less resistant to nisin A than the wild-type strain. A wild-type immunity level to nisin and production of nisin was obtained in strain NZ9800 harboring complementing nisA and nisZ plasmids. Transcription analyses of several L. IIactis strains indicated that an expression product of the nisA gene, together with NisR, is required for the activation of nisA transcription.

Selection for specific sequences in the external envelope protein of human immunodeficiency virus type 1 upon primary infection.

Abstract: Viral RNA was extracted from plasma samples collected from five individuals during the period of viremia before seroconversion in primary infection with human immunodeficiency virus type 1 (HIV-1) and amplified by polymerase chain reaction. Nucleotide sequence analysis of amplified DNA from the V3 and V4 hypervariable regions indicated that the initial virus population of each acutely infected individual was completely homogeneous in sequence. No intrasample variability was found among the 44,090 nucleotides sequenced in this region of env, contrasting with the high degree of variability normally found in seropositive individuals. Paradoxically, substantial sequence variability was found in the normally high conserved gag gene (encoding p17) in most of the preseroconversion samples. The diversity of p17 sequences in samples that were homogeneous in V3 and V4 can most readily be explained by the existence of strong selection for specific env sequences either upon transmission or in the interval between exposure and seroconversion in the exposed individual. Evidence that localizes the selected region upon transmission to V3 is provided by the similarity or identity of V3 loop sequences in five individuals with epidemiologically unrelated HIV-1 infections, while regions flanking the V3 loop and the V4 hypervariable region were highly divergent. The actual V3 sequences were similar to those associated with macrophage tropism in primary isolates of HIV, irrespective of whether infection was acquired by sexual contact or parenterally through transfusion of contaminated factor VIII. Proviral DNA sequences in peripheral blood mononuclear cells remained homogeneous in the V3 and V4 regions (and variable in p17gag) for several months after seroconversion. The persistence of HIV sequences in peripheral blood mononuclear cells identical to those found at primary infection in the absence of continued virus expression provides an explanation for the previously observed differences in the composition of circulating DNA and RNA populations in sequential samples from seropositive individuals.

Molecular cloning and characterization of acrA and acrE genes of Escherichia coli.

Abstract: The DNA fragment containing the acrA locus of the Escherichia coli chromosome has been cloned by using a complementation test. The nucleotide sequence indicates the presence of two open reading frames (ORFs). Sequence analysis suggests that the first ORF encodes a 397-residue lipoprotein with a 24-amino-acid signal peptide at its N terminus. One inactive allele of acrA from strain N43 was shown to contain an IS2 element inserted into this ORF. Therefore, this ORF was designated acrA. The second downstream ORF is predicted to encode a transmembrane protein of 1,049 amino acids and is named acrE. Genes acrA and acrE are probably located on the same operon, and both of their products are likely to affect drug susceptibilities observed in wild-type cells. The cellular localizations of these polypeptides have been analyzed by making acrA::TnphoA and acrE::TnphoA fusion proteins. Interestingly, AcrA and AcrE share 65 and 77% amino acid identity with two other E. coli polypeptides, EnvC and EnvD, respectively. Drug susceptibilities in one acrA mutant (N43) and one envCD mutant (PM61) have been determined and compared. Finally, the possible functions of these proteins are discussed.

At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative E1 gene of isolates collected worldwide

Abstract: In a previous study we sequenced the 5' noncoding (NC) region of 44 isolates of hepatitis C virus (HCV) and identified heterogeneous domains that provided evidence for additional genetic groups of HCV not previously recognized. In this study we have determined the complete nucleotide sequence of the putative envelope 1 (E1) gene in 51 HCV isolates from around the world and found that they could be grouped into at least 12 distinct genotypes. The E1 gene sequence of 8 of these genotypes has not been reported previously. Although the genetic relatedness of HCV isolates determined by the previous analysis of the 5' NC region predicted the relationships observed in the E1 gene, analysis of the 5' NC sequence alone did not accurately predict all HCV genotypes. The nucleotide and amino acid sequence identities of the E1 gene among HCV isolates of the same genotype were in the range of 88.0-99.1% and 89.1-98.4%, respectively, whereas those of HCV isolates of different genotypes were in the range of 53.5-78.6% and 49.0-82.8%, respectively. The latter differences are similar to those found when comparing the envelope gene sequences of the various serotypes of the related flaviviruses as well as other RNA viruses. We found that some genotypes of HCV were widely distributed around the world, whereas others were identified only in discreet geographical regions. Four genotypes were identified exclusively in Africa and comprised the majority of HCV isolates on that continent. The E1 gene was exactly 576 nucleotides in length in all 51 HCV isolates with no in-frame stop codons. Analysis of the predicted E1 protein identified several conserved domains that may be important for maintaining its biological function: (i) eight invariant cysteine residues, (ii) three potential N-linked glycosylation sites, (iii) a domain of nine amino acids (GHRMAWDMM), and (iv) an amino acid doublet (GV) near the putative cleavage site at the C terminus of the protein. In conclusion, the discovery of at least 12 genotypes of HCV has important implications for HCV diagnosis and vaccine development.

Reclassification of American Rhizobium leguminosarum biovar phaseoli type I strains as Rhizobium etli sp. nov.

Abstract: A new Rhizobium species that nodulates Phaseolus vulgaris L. is proposed on the basis of a sequence analysis of 16S ribosomal DNA. This taxon, Rhizobium etli sp. nov., was previously named Rhizobium leguminosarum biovar phaseoli (type I strains) and is characterized by the capacity to establish an effective symbiosis with bean plants, the reiteration of the nitrogenase structural genes, the organization of the common nodulation genes into two separate transcriptional units bearing nodA and nodBC, the presence of the polysaccharide inhibition gene, psi, and the 16S ribosomal DNA sequence. An analysis of the sequence of a fragment of the 16S rRNA gene shows that this gene is quite different from the gene of R. leguminosarum. In addition, all R. etli strains have identical sequences. We describe these analyses and discuss additional evidence supporting our proposal.

Reclassification of American Rhizobium leguminosarum Biovar Phaseoli Type I Strains as

Abstract: A new Rhizobium species that nodulates Phaseolus vulgaris L. is proposed on the basis of a sequence analysis of 16s ribosomal DNA. This taxon, Rhizobium etli sp. nov., was previously named Rhizobium leguminosarum biovar phaseoli (type I strains) and is characterized by the capacity to establish an effective symbiosis with bean plants, the reiteration of the nitrogenase structural genes, the organization of the common nodulation genes into two separate transcriptional units bearing nod4 and nodBC, the presence of the polysaccharide inhibition gene, psi, and the 16s ribosomal DNA sequence. An analysis of the sequence of a fragment of the 16s rRNA gene shows that this gene is quite different from the gene of R. leguminosarum. In addition, all R. etli strains have identical sequences. We describe these analyses and discuss additional evidence supporting our proposal. The bacteria of the genus Rhizobiurn nodulate the roots of leguminous plants. Rhizobiurn species have been defined primarily on the basis of phenotypic characteristics such as host range, colony morphology, growth on selective media, and certain metabolic attributes. The bacteria that infect peas, clovers, and beans (Phaseolus vulgaris L.) have been clustered in a single species, Rhizobiurn legurninosarurn, which has three biovars (I?. legurninosarurn biovar viciae, R. legurninosarurn biovar trifolii, and R. legurninosarurn biovar phaseoli) (11). This subdivision is based mainly on the presence of different symbiotic plasmids coding for distinct nodulation specificities in a uniform chromosomal background. The uniformity of this background has been supported by different types of data; the symbionts of peas, clovers, and beans cannot reliably be distinguished on the basis of phenotypic characteristics (17). Under laboratory conditions, recombination of chromosomal genes among isolates belonging to the different biovars can be obtained easily (9, 13); symbiotic plasmids conferring different symbiotic capacities can be transferred among strains isolated from peas, clovers, and beans and remain functional (7, 10, 14). DNA-DNA hybridization levels are higher among isolates belonging to the three biovars than to isolates belonging to other related species (3); the nucleotide sequences of a 16s rDNA fragment from different isolates obtained from peas, clovers, and beans have been found to be identical (6, 25). This classification has been challenged by several lines of evidence. Heterogeneity in R. legurninosarurn biovar phaseoli has been identified by a variety of methods, including protein patterns in two-dimensional polyacrylamide gel electrophoresis (21), antibiotic resistance profiles, serological types, DNA-DNA hybridization data (8), plasmid profiles, and exopolysaccharide structures (16). The genetic structures of several R. leguminosarurn populations have been determined. Young’s analysis of three enzymes in a collection of strains belonging to the three biovars of R. legurninosarurn isolated in English fields showed that there is a considerable amount of sharing of enzyme mobility variants

Identification and genetic analysis of normal and mutant phytoene synthase genes of tomato by sequencing, complementation and co-suppression

Abstract: A tomato phytoene synthase gene, Psyl, has recently been isolated as the clone GTOM5 [20] and shown by sequence identity to be the gene from which the major fruit-ripening cDNA clone TOM5 [19] was derived. Sequence analysis of transcripts from two allelic yellow-fruited tomato mutants, mapped to chromosome 3, has shown the lack of carotenoids in fruit of these mutants to be due to the production of aberrant TOM5 transcripts which are unlikely to encode a functional phytoene synthase enzyme. In one mutant (yellow flesh) the aberrant transcript contained a sequence that, by its strong hybridization to a wide size range of genomic fragments, appeared to be repeated many times within the genome. Southern and PCR analysis of the phytoene synthase genes in the mutant revealed restriction fragment length polymorphisms, suggesting that the production of altered mRNAs was associated with specific genomic rearrangements. Constitutive over-expression of a TOM5 cDNA clone in transgenic mutant plants restored synthesis of the carotenoid lycopene in ripening fruit and also led to unscheduled pigment production in other cell types. In some mutant plants transformed with the TOM5 cDNA construct, inhibition of carotenoid production in immature green fruit, leaves and flowers was observed, due to the phenomenon of co-suppression, indicating that different insertion events with the same gene construct can lead to overexpression or co-suppression in transgenic plants. Green organs of these plants were susceptible to photobleaching, due to the lack of carotenoids. These results suggest the existence of separate Psy genes for carotenoid synthesis in green organs.

The PROSITE dictionary of sites and patterns in proteins, its current status.

Abstract: PROSITE is a compilation of sites and patterns found in protein sequences; it can be used as a method of determining the function of uncharacterized proteins translated from genomic or cDNA sequences. In some cases the sequence of an unknown protein is too distantly related to any protein of known structure to detect its resemblance by overall sequence alignment, but relationships can be revealed by the occurrence in its sequence of a particular cluster of residue types which is variously known as a pattern, motif, signature, or fingerprint. These motifs arise because specific region(s) of a protein which may be important, for example, for their binding properties or for their enzymatic activity are conserved in both structure and sequence. These structural requirements impose very tight constraints on the evolution of these small but important portion(s) of a protein sequence. The use of protein sequence patterns to determine the function of proteins is becoming very rapidly one of the essential tools of sequence analysis. This reality has been recognized by many authors [1,2]. While there have been a number of reviews of published patterns [3,4,5], no attempt had been made until very recently [6,7] to systematically collect biologically significant patterns or to discover new ones. Based on these observations, we decided in 1988, to actively pursue the development of a database of patterns which would be used to search against sequences of unknown function. This database, called PROSITE, contains some patterns which have been published in the literature, but the majority have been developed in the last four years by the author.

Molecular analysis of spv virulence genes of the salmonella virulence plasmids

Abstract: Genes on an 8 kb region common to the virulence plasmids of several serovars of Salmonella are sufficient to replace the entire plasmid in enabling systemic infection in animal models. This virulence region encompasses five genes which previously have been designated with different names from each investigating laboratory. A common nomenclature has been devised for the five genes, i.e. spv for salmonella plasmid virulence. The first gene, spvR, encodes a positive activator for the following four genes, spvABCD. DNA sequence analysis of the spv genes from Salmonella typhimurium, Salmonella dublin, and Salmonella choleraesuis demonstrated extremely high conservation of the DNA and amino acid sequences. The spv genes are induced at stationary phase and in carbon-poor media, and optimal expression is dependent on the katF locus. The virulence functions of the spv genes are not known, but these genes may increase the growth rate of salmonellae in host cells and affect the interaction of salmonellae with the host immune system.

Structure and sequence relationships in the lipocalins and related proteins.

Abstract: The lipocalins and fatty acid-binding proteins (FABPs) are two recently identified protein families that both function by binding small hydrophobic molecules. We have sought to clarify relationships within and between these two groups through an analysis of both structure and sequence. Within a similar overall folding pattern, we find large parts of the lipocalin and FABP structures to be quantitatively equivalent. The three largest structurally conserved regions within the lipocalin common core correspond to characteristic sequence motifs that we have used to determine the constitution of this family using an iterative sequence analysis procedure. This afforded a new interpretation of the family, which highlighted the difficulties of determining a comprehensive and coherent classification of the lipocalins. The first of the three conserved sequence motifs is also common to the FABPs and corresponds to a conserved structural element characteristic of both families. Similarities of structure and sequence within the two families suggests that they form part of a larger "structural superfamily"; we have christened this overall group the calycins to reflect the cup-shaped structure of its members.

Mitochondrial DNA sequence analysis of human skeletal remains : Identification of remains from the Vietnam war

Abstract: Deoxyribonucleic acid (DNA) sequence analysis of the control region of the mitochondrial DNA (mtDNA) genome was used to identify human skeletal remains returned to the United States government by the Vietnamese government in 1984. The postmortem interval was thought to be 24 years at the time of testing, and the remains presumed to be an American service member. DNA typing methods using nuclear genomic DNA, HLA-DQ alpha and the variable number of tandem repeat (VNTR) locus D1S80, were unsuccessful using the polymerase chain reaction (PCR). Amplification of a portion of the mtDNA control region was performed, and the resulting PCR product subjected to DNA sequence analysis. The DNA sequence generated from the skeletal remains was identical to the maternal reference sequence, as well as the sequence generated from two siblings (sisters). The sequence was unique when compared to more than 650 DNA sequences found both in the literature and provided by personal communications. The individual sequence polymorphisms were present in only 23 of the more than 1300 nucleotide positions analyzed. These results support the observation that in cases where conventional DNA typing is unavailable, mtDNA sequencing can be used for human remains identification.

RNA editing in Newcastle disease virus.

Abstract: The co-transcriptional editing of the Newcastle disease virus (NDV) P gene has been studied by sequence analysis of cloned viral genomic RNA and mRNA. Evidence has been obtained for the specific insertion of non-templated G nucleotides, the consequence of which is the generation of three populations of P gene-derived mRNAs. The three populations encode proteins (P, V and W) which have a common N-terminal region, but which utilize three different reading frames at their C termini. Paradoxically, NDV edits its P gene mRNA by the insertion of non-templated G residues in a manner similar to Sendai and measles viruses (P → V editing) despite its apparent closer evolutionary relationship to the simian virus type 5, mumps and related group of viruses which edit a V genomic sequence to generate an mRNA to encode a functional P protein (V → P editing).

Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity.

Abstract: Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.

Genes on chromosomes 4, 9, and 19 involved in 11q23 abnormalities in acute leukemia share sequence homology and/or common motifs.

Abstract: Chromosome translocations involving band 11q23 are associated with human acute leukemias. These translocations fuse the ALL-1 gene, homolog of Drosophila trithorax and located at chromosome band 11q23, to genes from a variety of chromosomes. We cloned and sequenced cDNAs derived from transcripts of the AF-4 and AF-9 genes involved in the most common chromosome abnormalities, t(4:11)(q21:q23) and t(9:11)(p22:q23), respectively. Sequence analysis indicates high homology between the AF-9 gene protein product and the protein encoded by the ENL gene fused to ALL-1 in (11:19) chromosome translocations. AF-4, AF-9, and ENL proteins contain nuclear targeting sequences as well as serine-rich and proline-rich regions. Stretches abundant in basic amino acids are also present in the three proteins. These results suggest that the different proteins fused to ALL-1 polypeptide(s) provide similar functional domains.

Protein Ladder Sequencing

Abstract: A new approach to protein sequencing is described It consists of two steps: (i) ladder-generating chemistry, the controlled generation from a polypeptide chain by wet chemistry of a family of sequence-defining peptide fragments, each differing from the next by one amino acid; and (ii) data readout, a one-step readout of the resulting protein sequencing ladder by matrix-assisted laser-desorption mass spectrometry Each amino acid was identified from the mass difference between successive peaks, and the position in the data set defined the sequence of the original peptide chain This method was used to directly locate a phosphoserine residue in a phosphopeptide The protein ladder sequencing method lends itself to very high sample throughput at very low per cycle cost

Bacterial diversity in a soil sample from a subtropical Australian environment as determined by 16S rDNA analysis

Abstract: In order to investigate the genetic diversity of streptomycetes in an acid forested soil sample from Mt. Coot-tha, Brisbane, Australia, cells were mechanically lysed within the soil matrix and genomic DNA was isolated and purified. 16S ribosomal (r)DNA was amplified by the polymerase chain reaction (PCR) method using one primer conserved for members of the domain Bacteria and a second designed specifically for streptomycetes and related taxa. PCR amplification products were cloned into phage vector M13 mp19 and the diversity of 16S rDNA genes was determined by sequence analysis and oligonucleotide probing of the resultant clone library. Comparison of partial 16S rDNA sequences with published sequences revealed that few sequences originated from streptomycetes. The majority of sequences belonged to members of the alpha subclass of Proteobacteria. Other clones were related to planctomycetes, actinomycetes, or represented novel lines of descent. Bacteria that are customarily isolated from soil of pH 4-7 such...

Steroidogenic factor 1, an orphan nuclear receptor, regulates the expression of the rat aromatase gene in gonadal tissues.

Abstract: In a concerted analysis of the genes encoding three mouse steroid hydroxylases, we identified and characterized a transcriptional regulatory protein, designated steroidogenic factor 1 (SF-1), that contributes to the coordinate expression in adrenocortical cells. SF-1, an orphan member of the nuclear receptor family, binds to PyCAAGGPyCPu motifs upstream of the steroid hydroxylases to regulate their expression. In the present study, we extend these findings by examining the role of SF-1 in regulation of the rat P450 aromatase gene in gonadal tissues. The 5'-flanking region of the rat aromatase gene was isolated by a polymerase chain reaction-based approach, using primers corresponding to the 5'- and 3'-ends of a published aromatase sequence. DNA sequence analysis revealed three differences between our sequence and the previously published sequence, including a 44-base pair (bp) insertion. Moreover, the transcription initiation site, as determined by primer extension analysis, differed from that previously ...

Classification of plant-pathogenic mycoplasma-like organisms using restriction-site analysis of PCR-amplified 16S rDNA

Abstract: Summary: A method has been developed to amplify the 16S rRNA gene of plant-pathogenic mycoplasma-like organisms (MLOs) from infected plant material using the polymerase chain reaction (PCR). The procedure is dependent on the presence of a BclI restriction site in the 16S rDNA of chloroplasts but not in that of the MLOs. This difference permits the specific amplification of the 16S rDNA of the MLOs from BclI-digested total DNA from infected plants using primers from conserved regions of this gene. In this study 16S rDNA was obtained from 52 MLO isolates from herbaceous dicots and monocots as well as woody plants. Digestion of the 16S rRNA genes using AluI endonuclease revealed seven restriction patterns, which were used to group the isolates examined. Group I, which is also characterized by the presence of two KpnI sites, consisted of 31 isolates, most of which are from herbaceous dicots. Isolates assigned to groups II to VI were mostly from woody plants, while the isolates of group VII were from monocots or obtained from a leafhopper. The restriction patterns varied little within groups; however, four group I isolates and one group IV isolate differed slightly from the typical patterns of these groups as a result of a deletion or a slight shift of one restriction site. The groupings uncovered by AluI restriction were also obtained by digesting the 16S rDNA with RsaI endonuclease. However, some atypical patterns were observed within group V isolates. The groups described on the basis of restriction digest data were supported by sequence analysis. With one exception, the 16S rDNA of isolates within the same group exhibited 97·8 to 99·5% homology while those of different groups showed 89·6 to 92·0% homology.

Molecular genetic basis of the human Rhesus blood group system.

Abstract: The Rhesus (RH) blood group locus is composed of two related structural genes, D and CcEe, that encode red cell membrane proteins carrying the D, Cc and Ee antigens. As demonstrated previously, the RhD–positive/RhD–negative polymorphism is associated with the presence or the absence of the D gene. Sequence analysis of transcripts and genomic DNA from individuals that belong to different Rh phenotypes were performed to determine the molecular basis of the C/c and E/e polymorphisms. The E and e alleles differ by a single nucleotide resulting in a Pro226Ala substitution, whereas the C and c alleles differ by six nucleotides producing four amino acid substitutions Cys16Trp, He60Leu, Ser68Asn and Ser103Pro. With the recent cloning of the RhD gene, these findings provide the molecular genetic basis that determine D, C, c, E and e specificities.

Computer-assisted identification of a putative methyltransferase domain in NS5 protein of flaviviruses and lambda 2 protein of reovirus.

Abstract: A sequence motif that is conserved in a number of S-adenosylmethionine (SAM)-utilizing methyltransferases and is implicated in SAM binding was identified in the N-terminal portion of NS5 proteins of flaviviruses and in lambda 2 protein of reovirus. An additional conserved motif was shared by these viral proteins and two distinct groups of methyltransferases including as the prototypes Rhodobacter capsulatus hydroxyneurosporene methylase (crtF gene product) and yeast 3,4-dihydroxy-5-hexaprenylbenzoate methylase (COQ3 gene product), respectively. Statistically significant similarity was revealed between the region of flavivirus NS5 containing the SAM-binding motif and a newly characterized family of putative methyltransferases from bacteria, yeast and plants, which is related to the Coq3 group. Amino acid sequence signatures were derived that are unique for NS5 proteins and different subsets of (putative) cellular methyltransferases. It is hypothesized that the N-terminal domain of NS5 is a methyltransferase involved in viral RNA capping. Thus NS5 may be a two-domain protein, with its C-terminal domain comprising the RNA-dependent RNA polymerase. The putative methyltransferase domain of flaviviruses is unrelated to the methyltransferase domain previously characterized in positive-strand RNA viruses of the alphavirus-like supergroup. The lack of sequence similarity and different location of the putative methyltransferase domain underscores the drastic difference in the genome layout of flaviviruses and alphaviruses. The identification of the putative methyltransferase domain in reovirus lambda 2 protein is compatible with the available evidence that this protein is the viral capping enzyme.

Identification of a new class of nitrogen fixation genes in Rhodobacter capsulatus: a putative membrane complex involved in electron transport to nitrogenase

Abstract: DNA sequence analysis of a 12236 bp fragment, which is located upstream of nifE in Rhodobacter capsulatus nif region A, revealed the presence of ten open reading frames With the exception of fdxC and fdxN, which encode a plant-type and a bacterial-type ferredoxin, the deduced products of these coding regions exhibited no significant homology to known proteins Analysis of defined insertion and deletion mutants demonstrated that six of these genes were required for nitrogen fixation Therefore, we propose to call these genes rnfA, rnfB, rnfC, rnfD, rnfE and rnfF (for Rhodobacter nitrogen fixation) Secondary structure predictions suggested that the rnf genes encode four potential membrane proteins and two putative iron-sulphur proteins, which contain cysteine motifs (C-X2-C-X2-C-X3-C-P) typical for [4Fe--4S] proteins Comparison of the in vivo and in vitro nitrogenase activities of fdxN and rnf mutants suggested that the products encoded by these genes are involved in electron transport to nitrogenase In addition, these mutants were shown to contain significantly reduced amounts of nitrogenase The hypothesis that this new class of nitrogen fixation genes encodes components of an electron transfer system to nitrogenase was corroborated by analysing the effect of metronidazole Both the fdxN and rnf mutants had higher growth yields in the presence of metronidazole than the wild type, suggesting that these mutants contained lower amounts of reduced ferredoxins

Identification of repeat sequence heterogeneity at the polymorphic short tandem repeat locus HUMTH01[AATG]n and reassignment of alleles in population analysis by using a locus-specific allelic ladder.

Abstract: An allelic ladder containing amplified sequences of seven alleles of the polymorphic human tyrosine hydroxylase locus, HUMTH01, was constructed and employed as a standard marker. Sequence analysis of each ladder component indicates that fragments differ by integral multiples of the AATG core repeat sequence characteristic of this locus. Individual alleles are designated "5" through "11," according to the number of complete reiterations of the core repeat contained within them. Comparison of the HUMTH01 allelic ladder with DNA samples amplified at this locus revealed core repeat length heterogeneity (i.e., deletions or insertions shorter than one core repeat) within the human population. In particular, a common allele was identified which migrates more quickly than allele 10, but more slowly than allele 9, on electrophoresis through a denaturing polyacrylamide gel. Sequence analysis of this allele, designated "10-1," reveals lack of a single adenine normally present in the seventh copy of the AATG. The allelic ladder was used to reevaluate previously published population data. Results of testing for Hardy-Weinberg equilibrium and population substructure were not altered significantly by these modifications.