Showing papers on "Sequence analysis published in 1991"

DNA sequence analysis of NKG2, a family of related cDNA clones encoding type II integral membrane proteins on human natural killer cells.

Abstract: We have previously described the isolation of a cDNA clone, designated NKG2, that was expressed in all natural killer (NK) cells tested but not in T or B cells. In the present communication, the original isolate, when used to probe a cDNA library prepared from a CD3- NK cell clone, was found to crosshybridize with a family of transcripts that fell into four distinct groups designated NKG2-A, -B, -C, and -D. Full-length cDNA sequences were determined for each group, and the DNA and inferred peptide sequences were analyzed. All four transcripts encode type II membrane proteins of 215-233 amino acids. NKG2-A and -B peptides appear to be alternative splicing products of a single gene. NKG2-C is highly homologous with group A, having 94% homology in the external (COOH-terminal) domain and 56% homology throughout the internal and transmembrane regions. NKG2-D is distantly but significantly related (21% amino acid homology) to the first three groups. Therefore, NKG2-A, -C, and -D appear to be encoded by distinct genes within a family of NK cell-specific genes. Peptide sequence homology searches demonstrate that the NKG2 peptides are members of a supergene family that includes several other type II membrane proteins. This family is characterized by the presence of a C-type animal lectin domain, and several of its members have demonstrated transmembrane signaling capability.

The 16s/23s ribosomal spacer region as a target for DNA probes to identify eubacteria.

Abstract: Variable regions of the 16s ribosomal RNA have been frequently used as the target for DNA probes to identify microorganisms. In some situations, however, there is very little sequence variation observed between the 16s rRNA genes of closely related microorganisms. This study presents a general method to obtain species-specific probes using the spacer (intergenic) region between the 16s and 23s rRNA genes. The overall strategy is analogous to that which has previously been developed for the variable regions of the 16s rRNA genes. Sequence analysis of the 16s rRNA and 23s rRNA coding sequences flanking the spacer regions resulted in the design of PCR primers that can be used to amplify the spacer regions of a wide range of eubacteria. Sequencing the amplified spacer region then gives rise to the information that can be used to select specific DNA sequences for use as a DNA probe or for the generation of specific PCR primers to a microorganism of interest. In this study the approach to develop specific DNA markers for members of the genus Clostridium is described in detail. A specific DNA oligonucleotide probe and PCR primers have been designed for Clostridium perfringens that distinguish it from other organisms in the genus.

Phylogenetic Analysis of DNA Sequences

Abstract: 1. Phylogenetic inference, DNA sequence analysis, and the future of molecular systematics 2. DNA sequencing: strategy and methods to directly sequence large DNA molecules 3. The application of automated DNA sequence analysis to phylogenetic studies 4. Computer alignment of sequences 5. Aligning DNA sequences: homology and phylogenetic weighting 6. Relative efficiencies of different tree-making methods for molecular data 7. Compositional statistics evaluated by computer simulations 8. Weighted parsimony: does it work? 9. Testing the theory of descent 10. Parsimony and phylogenetic inference using DNA sequences: some methodological strategies 11. Evolutionary analysis of length-variable sequences: divergent domains of robosomal RNA 12. Statistical methods for testing molecular phylogenies 13. Discriminating between phylogenetic signal and random noise in DNA sequences 14. When are phylogeny estimates from morphological and molecular data incongruent? 15. Congruence among data sets: a Bayesian approach

Differentially expressed isoforms of the mouse retinoic acid receptor beta generated by usage of two promoters and alternative splicing.

Abstract: Using anchored PCR, three different cDNA isoforms of the mouse retinoic acid receptor beta [mRAR-beta 1, mRAR-beta 2 (formerly mRAR-beta 0) and mRAR-beta 3], generated from the same gene by differential promoter usage and alternative splicing, were isolated. These three isoforms encode RAR proteins with different N-terminal A regions and identical B - F regions. The sequence encoding the first 59 amino acids of the mRAR-beta 3 A region is identical with the entire A region of mRAR-beta 1. However, the sequence of mRAR-beta 3 region A differs from that of mRAR-beta 1 by an additional 27 C-terminal amino acids encoded in an 81 nucleotide-long putative exon which is spliced in between the exons encoding the A and B regions of mRAR-beta 1. Both mRAR-beta 1 and beta 3 cDNAs differ entirely from mRAR-beta 2 in their 5'-untranslated (5'-UTR) and A region coding sequences. This N-terminal variability, in a region which was shown to be important for cell-type specific differential target gene trans-activation by other nuclear receptors, suggests that the three mRAR-beta isoforms may be functionally distinct. The conservation of RAR-beta isoform sequences from mouse to human, as seen by cross-hybridization on Southern blots or DNA sequence analysis, as well as their differential patterns of expression in various mouse tissues, corroborates this view. Additionally, the mRNA analysis data suggest that mRAR-beta 2, whose expression predominates in RA-treated embryonal carcinoma (EC) and embryonic stem (ES) cells, may be important during early stages of development. mRAR-beta 1 and beta 3, on the other hand, which are predominantly expressed in fetal and adult brain, may play some specific role in the development of the central nervous system.

A distinct potassium channel polypeptide encoded by the Drosophila eag locus.

Abstract: Many of the signaling properties of neurons and other electrically excitable cells are determined by a diverse family of potassium channels. A number of genes that encode potassium channel polypeptides have been cloned from various organisms on the basis of their sequence similarity to the Drosophila Shaker (Sh) locus. As an alternative strategy, a molecular analysis of other Drosophila genes that were defined by mutations that perturb potassium channel function was undertaken. Sequence analysis of complementary DNA from the ether a go-go (eag) locus revealed that it encodes a structural component of potassium channels that is related to but is distinct from all identified potassium channel polypeptides.

Cloning and characterization of cDNA of avirulence gene avr9 of the fungal pathogen Cladosporium fulvum, causal agent of tomato leaf mold.

Abstract: A race-specific peptide elicitor from Cladosporium fulvum induces a hypersensitive response on Cf9 tomato genotypes. We have hypothesized that the avirulence of fungal races on Cf9 genotypes is due to the production of this elicitor by an avirulence gene, avr9. To obtain cDNA clones of the avr9 gene, oligonucleotide probes were designed based on the amino acid sequence determined previously. In northern blot analysis, one oligonucleotide detected an mRNA of 600 nucleotides in tomato-C. fulvum interactions involving fungal races producing the elicitor. A primer extension experiment indicated that the probe hybridized to a region near position 270 of the mRNA. The probe was used to screen a cDNA library made from poly(A)+ RNA from an appropriate compatible tomato-C. fulvum interaction. One clone was obtained corresponding to the mRNA detected by the oligonucleotide probe. Sequence analysis revealed that this clone encoded the avr9 elicitor. By isolating longer clones and by RNA sequencing, the primary structure of the mRNA was determined. The mRNA contains an open reading frame of 63 amino acids, including the sequence of the elicitor at the carboxyterminus. A time course experiment showed that the avr9 mRNA accumulates in a compatible tomato-C. fulvum interaction in correlation with the increase of fungal biomass. The avr9 gene is a single-copy gene that is absent in fungal races which are virulent on tomato Cf9 genotypes. Possible functions of the avirulence gene are discussed.

Analysis of Interstrain Variation in Cytomegalovirus Glycoprotein B Sequences Encoding Neutralization-Related Epitopes

Abstract: Nucleotide sequences of a part of the envelope glycoprotein B (gB) gene of human cytomegalovirus (CMV), encoding epitopes recognized by virus-neutralizing monoclonal antibodies, were determined for 12 distinct clinical strains of CMV after amplification of suitable templates using the polymerase chain reaction. Sequence analysis of this region (codons 384-717) revealed that the clinical strains and previously sequenced laboratory strains Towne and AD169 belong to one of four variant groups, each with a characteristic nucleotide and peptide sequence. Peptide homology was greater than 99% for strains within a group, and varied from 91% to 98% for strains in different groups. Variation was most frequent between codons 448 and 480. The gB group of a CMV strain could be determined by restriction analysis of a small target sequence amplified from viral genomic DNA, and an additional 28 clinical strains were grouped in this manner. The existence of a limited number of variants of gB among clinical strains facilitates analysis of biologic function and cross-reactivity of immune responses.

Identification of mRNAs associated with programmed cell death in immature thymocytes.

Abstract: Programmed cell death is an essential cellular process that occurs in epithelial turnover, neural development, and regulation of cell populations of the immune system Thymocytes undergo programmed cell death in response to several inductive stimuli, including exposure to glucocorticoids or radiation This program can be blocked by inhibitors of RNA or protein synthesis; this implies that new proteins are required to execute the death programs To search for possible death-associated mRNAs, we directionally cloned cDNA representing mRNA from control and dexamethasone-treated thymocytes These libraries were used to produce ample amounts of DNA and RNA used in subtractive hybridization for the removal of sequences present in both control and induced cells The remaining unhybridized sequences were selectively amplified by polymerase chain reaction and cloned to produce a library enriched for sequences expressed in death-induced cells From this library we isolated cDNAs of death-associated mRNAs One of these mRNAs, RP-8, appears within 1 h after exposure to gamma radiation, and a second mRNA, RP-2, is observed within 2 h Both of these mRNAs accumulate during a period when a reference mRNA, actin, is declining RP-2 and RP-8 are no longer detectable after 6 h postinduction, when apoptosis and mRNA degradation are evident in the culture Sequence analysis of RP-8 cDNA indicates the presence of a zinc finger domain suggestive of a possible DNA regulatory role for the RP-8 protein cDNA sequence results on RP-2 classify the corresponding protein as an integral membrane protein We conclude that RP-2 and RP-8 are death-associated mRNAs that should be functionally evaluated in the context of the death process As previously suggested, it may be that a family of "death genes" is activated by various stimuli depending on the type of cell, in a manner somewhat analogous to the induction of heat shock (stress) protein genes

Herpes simplex virus type 1 deletion variants 1714 and 1716 pinpoint neurovirulence-related sequences in Glasgow strain 17+ between immediate early gene 1 and the 'a' sequence.

Abstract: Dideoxynucleotide sequence analysis of a spontaneously isolated deletion variant (1714) of Glasgow strain 17+ of herpes simplex virus type 1 (HSV-1) demonstrates that the deletion is 759 bp in length and is located within each copy of the BamHI s fragment (0 to 0.02 and 0.81 to 0.83 map units) of the long repeat region of the genome. The deletion removes one complete copy of the 18 bp DR1 element of the ‘a’ sequence and terminates 1105 bp upstream of the 5′ end of immediate early (IE) gene 1. The variant grows to high titre, is not temperature-sensitive and is not host cell type-restricted in vitro. In vivo studies demonstrate that 1714 is totally avirulent for BALB/c mice following intracerebral inoculation, with an LD50 of 7 × 106 p.f.u./mouse compared to < 10 p.f.u./mouse for the parental wild-type strain 17+. In vivo growth kinetics show that the non-neurovirulent phenotype is due to an inability to replicate in mouse brain. Because 1714 was in a genomic background in which the four XbaI sites had been removed and because the phenotype was thymidine kinase-negative, the 759 bp deletion was introduced into an otherwise totally wild-type background. The resulting variant (1716) is nonneurovirulent for mice, with an LD50 of 7 × 106 p.f.u./mouse. The deletion does not prevent the virus from establishing a latent infection or reactivating from it in vitro. The results demonstrate that sequences between IE-1 and the ‘a’ sequence produce neurovirulence in Glasgow strain 17+ and, in conjunction with the nonneurovirulence of the HSV-2 HG52 variant JH2604, identify a common function conserved in HSV-1 and -2.

Molecular cloning and sequence analysis of bovine lactotransferrin.

Abstract: The screening of a bovine submaxillary gland cDNA library yielded 25 clones coding for bovine lactotransferrin. The nucleotide sequence of the longest insert contained a protein-coding region of 2115 nucleotides and a 3′ non-coding region of 194 nucleotides followed by a poly(A) tract of about 55 nucleotides. The predicted peptide sequence included a 16-amino-acid signal sequence upstream of the first amino acid of the native protein. The identity of the clone was confirmed by matching the amino acid sequence predicted from the cDNA with the N-terminal and tryptic peptide sequences derived from purified bovine milk lactotransferrin, and also by similarity with human and murine lactotransferrins. The cDNA described corresponds to a 705-amino-acid-long preprotein that lacks the start methionine. The sequence of the secreted protein is 689 amino acids long and contains five potential glycosylation sites. Bovine lactotransferrin is 69% and 64% identical to human and murine lactotransferrins, respectively.

Sequence analysis of cDNA coding for a major house dust mite allergen, Der f I

Abstract: Summary A cDNA clone-coding for Der f I, a major allergen from the house dust mite Dermatophagoides farinae has been isolated and sequenced. It codes for a putative 18-residue signal peptide, an 80-residue proenzyme region, and a 223-residue mature protein with a derived molecular weight of 25 191. The deduced amino-acid sequence shows significant homology to other cysteine proteases in the proregion as well as in the mature protein. Sequence alignment of the mature Der f I protein with the homologous allergen Der p I from the related mite D. pteronyssinus revealed a high degree of homology (81%) between the two proteins, as predicted by previous sequencing at the protein level. In particular, the residues comprising the active site of these enzymes and the cysteine residues were conserved. A potential N-glycosylation site was present at an equivalent position in both mite allergens. It is anticipated that the availability of recombinant Derf I will facilitate epitope mapping studies and studies of T-cell function in mite allergy by providing high levels of pure allergen.

Identification of Thunnus Tuna Species by the Polymerase Chain Reaction and Direct Sequence Analysis of their Mitochondrial Cytochrome b Genes

Abstract: Four commercially important tuna species in the genus Thunnus are caught off the east coast of Canada. The harvest of bluefin tuna (T. thynnus) is regulated, but that of bigeye (T. obesus), yellowf...

Intrageneric Structure of Streptococcus Based on Comparative Analysis of Small-Subunit rRNA Sequences

Abstract: The partial 16S rRNA sequences of 24 Streptococcus species were determined by reverse transcription. A comparative analysis of these sequences and the sequences of seven previously studied streptococcal species revealed the presence of several clusters within the genus. The clusters obtained from the sequence analysis agreed in general with the groups outlined on the basis of the results of nucleic acid hybridization studies, but there were some exceptions. The pyogenic group was extended to include Streptococcus agalactiae, S. parauberis, S. porcinus, and S. uberis. Four oral groups were discerned; these four groups centered on S. mutans, S. salivarius, S. anginosus, and S. oralis. Some species (e.g., S. suis and S. acidominimus) did not cluster with any particular group. Our findings are discussed in the context of data from other genetic and chemotaxonomic studies.

Carrier detection and prenatal diagnosis in Duchenne and Becker muscular dystrophy families, using dinucleotide repeat polymorphisms

Abstract: To improve carrier detection and prenatal diagnosis for Duchenne and Becker muscular dystrophy families, we determined allele frequencies and measures of variation for four (dC-dA)n.(dG-dT)n loci identified within a deletion-prone region of the human dystrophin gene. The loci are highly polymorphic, with predicted heterozygosities of 71.6%-93.3%. Direct DNA sequence analysis of the (dC-dA)n.(dG-dT)n locus in intron 49 revealed an additional length polymorphism which varies by single-basepair increments, is adjacent to the dinucleotide repeat block, and enhances the polymorphic content of this marker. The four (dC-dA)n.(dG-dT)n loci are each easily amplified by PCR in two diplex reactions. The variability of allele lengths at these loci makes them ideal for carrier detection and prenatal diagnosis, often providing diagnostic information when RFLP analysis is uninformative. These markers have aided in identification of deletion mutations, exclusion of maternal cell contamination of chorionic villus samples, confirmation of paternity, and mapping of gene recombinations. The allele identification of these loci can be performed either with a radiolabel or with an automated, nonradioactive, fluorescent gel detection system.

Identification of potyviruses using the polymerase chain reaction with degenerate primers.

Abstract: Local areas of conserved amino acid sequence in the replicase and coat proteins of potyviruses were used to select nucleotide sequences for use in the construction of sets of degenerate oligonucleotide primers for amplification of DNA fragments on potyvirus-specific templates in a combined assay of reverse transcription and the polymerase chain reaction (RT-PCR). Sequences selected for the construction of degenerate primers included the coat protein gene sequence of tulip breaking virus from lily, which is reported in this paper. It is shown that the degenerate primers support potyvirus-specific amplification, but do not support amplification on carlavirus and potexvirus templates. A panel consisting of definite and prospective members of the potyvirus group occurring in bulbous crops was subjected to the degenerate primer RT-PCR assay; amplified fragments were used in cross-hybridization experiments and restriction fragment length polymorphism analysis to detect relationships among these potyviruses. A partially characterized virus isolated from Gloriosa rothschildiana was positively identified as a potyvirus by specific amplification and subsequent sequence analysis of an amplified DNA fragment.

Amino acid sequence of Fel dI, the major allergen of the domestic cat: protein sequence analysis and cDNA cloning.

Abstract: The complete primary structure of Fel dI (International Union of Immunological Societies nomenclature), the major allergen produced by the domestic cat, Felis domesticus, was determined by protein sequence analysis and cDNA cloning. Protein sequencing of Fel dI from an immunoaffinity-purified extract of house dust revealed that the allergen is composed of two polypeptide chains. Degenerate oligonucleotides derived from the protein sequence were used in polymerase chain reaction amplification of cat salivary gland cDNA to demonstrate that the two chains are encoded by different genes. Chain 1 of Fel dI shares amino acid homology with rabbit uteroglobin, while chain 2 is a glycoprotein with N-linked oligosaccharides.

Structure of the 55-kDa regulatory subunit of protein phosphatase 2A: evidence for a neuronal-specific isoform

Abstract: The trimeric form of protein phosphatase 2A (PP2A1 or polycation-stimulated protein phosphatase H1) was purified to homogeneity from rabbit skeletal muscle. Preparative SDS-polyacrylamide gel electrophoresis was used to purify the individual subunits with relative molecular masses of 36, 55, and 65 kDa. Sequence analysis of five peptides from the 65-kDa regulatory subunit (PR65) suggested that it was identical with the PR65 subunit derived from the dimeric protein phosphatase 2A2. Amino acid sequences derived from the 55-kDa regulatory subunit (PR55) were used to clone human and rabbit cDNAs encoding this protein. The PR55 subunit was found to be encoded by two genes, termed alpha and beta. The open reading frames of the PR55 alpha and beta cDNAs spanned 1341 and 1329 nucleotides, respectively, and predicted proteins with a molecular mass of about 52 kDa that are 86% identical. Comparison of the human PR55 amino acid sequences with the data obtained from the rabbit skeletal muscle protein and a partial rabbit PR55 beta cDNA clone indicated a high degree of conservation. Analysis of the mRNA expression in human cell lines revealed that the PR55 alpha isoform was encoded by two transcripts of about 2.3 and 2.5 kb and a less abundant 4.4-kb mRNA. Whereas a PR55 beta transcript of about 2.3 kb was detected at high levels in the neuroblastoma derived cell line LA-N-1, the level of the mRNA was very low in the other human cell lines analyzed. Interestingly, the PR55 sequence showed limited homology to the catalytic domain (domains VI-IX) of the c-abl protein tyrosine kinase.

LcrD, a membrane-bound regulator of the Yersinia pestis low-calcium response.

Abstract: Yersinia pestis, the etiologic agent of bubonic plague, contains a 75-kb virulence plasmid, called pCD1 in Y. pestis KIM. The low-Ca(2+)-response genes of Y. pestis regulate both bacterial growth and the expression of pCD1-encoded virulence determinants in response to temperature and the presence of Ca2+ or nucleotides. This study characterizes the nucleotide sequence and protein product of the lcrD locus. An lcrD mutant, in contrast to the parent Y. pestis, did not undergo growth restriction or induce strong expression of the V antigen when grown under conditions (37 degrees C, no Ca2+) expected to elicit maximal expression of pCD1 genes. DNA sequence analysis of the cloned lcrD locus showed a single open reading frame that could encode a protein with a molecular weight of 77,804 and a pI of 4.88. LcrD was identified as a 70-kDa inner membrane protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. LcrD membrane topology was investigated by using lcrD-phoA translational fusions generated with the transposon TnphoA. The alkaline phosphatase activities of the resultant hybrid proteins were consistent with a model predicting eight amino-terminal transmembrane segments that anchor a large cytoplasmic carboxyl-terminal domain to the inner membrane.

Sequence analysis and expression of the host-protective immunogen VP2 of a variant strain of infectious bursal disease virus which can circumvent vaccination with standard type I strains.

Abstract: The host-protective antigen VP2 of a variant strain of infectious bursal disease virus (IBDV) which emerged from a vaccinated flock and is able to circumvent vaccination with classic type I strains of IBDV, was cloned and its nucleotide sequence determined. Virus-neutralizing monoclonal antibodies (MAbs) raised against the Australian 002-73 strain of IBDV did not react or reacted only very weakly with the expression product of the variant virus. The deduced amino acid sequence of VP2 from the variant strain differed in 17 residues from that of the Australian strain and in eight positions from a consensus sequence compiled from six type I strains of IBDV. All the amino acid changes mapped within the central, variable region of VP2, which forms the conformational epitope recognized by virus-neutralizing MAbs. Changes in the two hydrophilic regions at either end of this fragment were unique to the variant virus and were crucial for its ability to escape the virus-neutralizing antibodies induced by vaccination with a standard type I vaccine.

Typing of hepatitis C virus genomes by restriction fragment length polymorphism.

Abstract: Recently, we reported that hepatitis C virus (HCV) can be classified genetically into two types, HCV-K1 and HCV-K2, which show 67% and 71% identity at the nucleotide and amino acid sequence levels in a 340 bp region which encodes the NS5 gene Gly-Asp-Asp motif. To develop a rapid method to classify the genomes of HCV isolates, we identified restriction fragment length polymorphisms (RFLPs) in reverse transcriptase-polymerase chain reaction products encoding a portion of the NS5 gene. AluI and AccII enabled HCV to be classified into the K1 and K2 types, and Sau96I enabled classification into the K1 type, and the K2a and K2b subtypes. These RFLPs also generally allow Japanese isolates to be distinguished from the prototype (PT, an isolate from the U.S.A.), which is a K1 type. Sequence analysis of the 5′-untranslated regions of Japanese isolates revealed near identity between the K1 type and PT, and 93 to 94% identity between the K1 and K2 types, indicating that there are type K1- and K2-specific RFLPs in this region. Our results suggest that the nucleotide sequences of the K1 and K2 types are different throughout the HCV genome. The incidence of HCV types K1, K2a and K2b, and PT in 50 samples was 74%, 16%, 8% and 2%, respectively.

A comparison of optimal and suboptimal RNA secondary structures predicted by free energy minimization with structures determined by phylogenetic comparison

Abstract: This article describes the latest version of an RNA folding algorithm that predicts both optimal and suboptimal solutions based on free energy minimization. A number of RNA's with known structures deduced from comparative sequence analysis are folded to test program performance. The group of solutions obtained for each molecule is analysed to determine how many of the known helixes occur in the optimal solution and in the best suboptimal solution. In most cases, a structure about 80% correct is found with a free energy within 2% of the predicted lowest free energy structure.