Structural Gene Sequences Research Articles

Nucleotide and deduced amino acid sequence of the structural protein genes of Japanese encephalitis viruses from different geographical locations

Strain variation among Japanese encephalitis (JE) virus isolates has been previously demonstrated by many workers using different methods. We report the nucleotide sequence of the 5' non-coding region and the nucleotide and deduced amino acid sequences of structural protein genes for eight wild-type JE virus strain isolated from different Asian countries (Vietnam, Nepal, Indonesia, Thailand, India, Japan and China). We compare these with five other published wild-type JE virus strains isolated from Japan and the mainland of China. No strain variation could be related to geographical location on the basis of the wild-type JE virus strains studied. The viruses differed from each other by between 21 (0.9%) and 111 (4.6%) nucleotides and by between 3 (0.4%) and 31 (4.2%) amino acids over the 2434 nucleotides examined. The amino acid divergence of the envelope (E) protein gene of the viruses was 4.2% or less among the 13 strains compared. Thus there is little genetic divergence in the strains studied. However, four variable E protein amino acids (E-51, E-209, E-244 and E-366) were identified. Residue positions E-51, E-244 and E-366 are found in peptides with functional T helper cell epitopes in two other flaviviruses. Therefore, these amino acids may be important in defining the various immunotypes of JE virus identified by antibody reactivity patterns.

The use of arms PCR and RFLP analysis in identifying genetic profiles of virulent, attenuated or vaccine strains of TGEV and PRCV.

The use of ARMS (amplification refractory mutation system) PCR coupled with RFLP (restriction fragment length polymorphism) analysis has been used to identify a unique genetic marker on the Ambico oral vaccine strain. This method was also used to characterize the genetic profiles of a number of other TGEV strains. This procedure takes advantage of the nucleotide differences between the Ambico strain, and the Miller and Purdue strains. Within the S gene there are three nucleotide differences between the Ambico strain and the published Purdue sequence. There are additional nucleotide differences in the structural and non-structural gene sequences, but we have chosen to focus on the differences contained within the S gene. The Ambico strain has a closer sequence homology to the Purdue strain than to the Miller strain. The Ambico and Purdue strains contain a six nucleotide deletion at position 1122 that is not present in the Miller published sequence or the ISU-1 strain of PRCV (based on our PCR experiments). We have designed a 5' oligo whose sequence is homologous to a region located 80 nucleotides upstream of the TGEV and PRCV S gene initiation codons to be used in conjunction with either of two 3' oligos whose sequences are identical with the exception of the last six nucleotides of their 3' ends. When utilized with the appropriate PCR conditions, these oligos can differentiate between PRCV, Miller and Purdue prototype virus strains. These PCR products were then subjected to RFLP analysis using four separate restriction enzymes (BstE II, Alw26 I, Dra III, or MspA1 I). We have used this procedure to analyze six TGEV vaccine strains, intestinal derived virulent viruses, cell cultured viruses at different cell passage numbers, and field isolates of TGEV or PRCV.

The identification of heterogeneity of 5-HT3 receptors with [3H]RS-42358-197.

The diverse pharmacological action of serotonin (5-HT) has been the subject of intense study since its identification in 1936.1 The diversity of actions include activation or inhibition of smooth and cardiac muscle, exocrine and endocrine glands, cells of the hematopoietic and immune systems, as well as central and peripheral neurons.2,3 The results of these investigations have led to the identification of at least 12 different 5-HT receptors based on operational (functional, antagonism, location), transductional (G-protein, ion channel), and structural (gene sequence, chromosomal location) criteria.4 Based on these criteria, 5-HT receptors have been grouped into 5 types, namely 5-HT1 5-HT2, 5-HT3, 5-HT4, and 5-HT5 receptors.5,6

Isolation of Chicken hsp90β Gene Promoter

In order to define the mechanisms responsible for the differential expression of chicken hsp90α and β genes, a portion of the chicken hsp90β genomic sequence, including 3081 bp upstream from the transcription initiation site and 2718 bp of structural gene sequence which covers 7 exons and 6 introns was investigated. The transcriptional initiation site was determined by primer extension, RNAase and S1 nuclease mapping, Northern blot and cloning of 5′ end of cDNA. The first intron, as in other hsp90 genes, is located just before the ATG initiation codon. Three Sp l sites are located near the TATA box. The apparent major divergence with the hsp90α promoter is that, in the hsp90β promoter, the only CAAT box and HSE element are located at about 3 and 2 kb upstream the TATA box,respectively. These features may explain why chicken hsp90β mRNA is generally less abundant than α and is not inducible by heat shock or serum/growth factor stimulation.

Detection of Pseudomonas aeruginosa from clinical and environmental samples by amplification of the exotoxin A gene using PCR.

PCR was used to detect Pseudomonas aeruginosa from water samples by amplifying a 396-bp region of the exotoxin A (ETA) structural gene sequence. The identify of the amplified 396-bp fragment was confirmed by digesting it with PvuI restriction endonuclease, which produced the predicted 246- and 150-bp fragments. Specific primers amplified ETA-positive P. aeruginosa DNA, whereas other species of Pseudomonas and GC-rich bacteria did not yield any 396-bp fragment. The specificity and sensitivity of the assay were 100 and 96%, respectively, which confirms the assay's reliability for diagnostic and epidemiological studies. The assay can detect as few as 5 to 10 cells in a 10-ml water sample or 0.1 pg of P. aeruginosa DNA per reaction mixture (5 microliters) by ethidium bromide staining of an agarose gel. Ten-times-lower concentrations were detected by hybridization with a digoxigenin-labeled oligonucleotide probe internal to the PCR product. With this PCR method, ETA-positive P. aeruginosa was detected in animal cage water samples at a level of 40 cells per ml. This method is rapid and less cumbersome than other diagnostic methods for the identification of P. aeruginosa strains. The method described can be used to detect a low level of P. aeruginosa from environmental and clinical samples without the use of selective media or additional biochemical tests.

Modern 5-HT receptor classification and 5-HT based drugs

Based on operational (agonist and antagonist rank order), transductional (second messenger coupling) and structural (gene and amino acid sequence) criteria, the Nomenclature Committee of the Serotonin Club has discerned four distinct 5-HT receptor types, besides two less well characterised categories including some recombinant and the so-called ‘orphan’ receptors. The responses mediated via many 5-HT receptors are now well understood and this, in turn, has resulted in the development and use of serotonergic drugs in the therapy of several diseases, including anxiety and migraine, and in the prevention of vomiting caused by anticancer agents. Many other therapeutic avenues are being explored presently.

Analysis of the structural protein gene sequence shows Kyasanur Forest disease virus as a distinct member in the tick-borne encephalitis virus serocomplex

Kyasanur Forest disease (KFD) virus is a highly pathogenic member of the family Flaviviridae producing a haemorrhagic disease in infected human beings. Despite this high pathogenicity and potential epidemiological importance, there have been relatively few detailed antigenic or molecular studies on KFD virus. The nucleotide sequences of the genes encoding the structural proteins of the virus have now been determined. From these data we conclude that KFD virus is a distinct member in the tick-borne flavivirus complex with characteristic protease cleavage sites, fusion peptide, signal sequences and hydrophobic transmembrane domains. Comparison of the deduced amino acid sequences of KFD virus showed close relationships with other tick-borne flaviviruses. Among the structural proteins, the E protein showed maximum similarity (77.4% to 81.3%) to tick-borne flaviviruses. Alignment of the amino acid sequence with those of other known tick-borne flaviviruses revealed many conserved regions confirming its identity as a member of the tick-borne encephalitis group, although the genetic marker EHLPTA showed a T-->K substitution in KFD virus. The proposed genetic marker at amino acid positions 232 to 234 (AQE) was unique for KFD virus. A dendrogram derived from the amino acid alignment showed a phylogenetic relationship similar to those obtained on the basis of serological studies. The question of the sudden emergence of KFD virus in India and the possibilities of developing recombinant virus vaccines are discussed.

Variation and Genetic Control of Surface Antigen Expression in Mycoplasmas: The VIp System of Mycoplasma hyorhinis

Surface antigenic diversity in the swine pathogen Mycoplasma hyorhinis is generated by random combinatorial expression and high-frequency phase variation of multiple, size-variant membrane surface lipoproteins (Vlps) which represent the major coat proteins of this wall-less procaryote. The distinctive structural basis for Vlp variation was revealed in a family of several related but divergent vlp genes. These occur in one cluster as single chromosomal copies, each encoding a conserved domain for membrane insertion and lipoprotein processing, and a divergent external domain that changes size by deletion or insertion of repetitive intragenic coding sequences while retaining a distinctive charge motif. Lack of detectable changes in restriction fragment patterns or DNA sequence of vlp structural genes during phase transitions between ON and OFF expression states ruled out long range genomic rearrangements and frameshift mutations as a means of controlling Vlp phase variation. However, highly homologous vlp promoter regions contain a homopolymeric tract of contiguous adenine residues [poly(A)] upstream of the transcriptional start site which is subject to frequent mutations altering its length. These mutations are the only sequence changes detected during phase transitions, and are highly correlated with the expression state of each vlp gene. This suggests a mechanism of transcriptional control regulating Vlp phase variation by critical changes within the poly(A) region affecting the spacing between the -10 and -35 hexamers or a putative regulator binding site. The multiple levels of structural and antigenic diversity embodied in the vlp gene family may provide essential adaptive capabilities for this wall-less microbial pathogen.

Evolution of structural proteins of feline immunodeficiency virus: molecular epidemiology and evidence of selection for change

The DNA sequences of structural genes of several U.K. and European isolates of feline immunodeficiency virus (FIV) were determined and compared with those of other worldwide isolates. Phylogenetic analyses of both gag and env sequences demonstrate that a Japanese isolate represents a distinct sequence subgroup, with corrected amino acid distances to the other isolates averaging 23% in env and 8% in gag. Analysis also reveals that an evolutionary radiation of FIV occurred with many isolates diverging at approximately the same time, and that although isolates from similar geographical sources often cluster together, there is evidence of more than one origin for FIV in the U.K., The Netherlands and Italy. Estimation of the numbers of silent and replacement nucleotide substitutions indicates the presence of constraints against amino acid changes in gag and conserved regions of env but suggests that positive selection for protein sequence changes operates in variable regions of env. The possible immunological forces underlying these changes are discussed.

Nucleotide sequence of the cynomolgus monkey apolipoprotein A-I gene and corresponding flanking regions

The cynomolgus monkey ( Macaca fascicularis) apolipoprotein A–I (apo A–I) gene and corresponding flanking regions have been isolated from a cynomolgus monkey genomic library and completely sequenced. Comparison with the human sequence shows a > 90% homology with the monkey sequence, overall. The monkey apo A–I structural gene (1856 bp) is representative of the general apolipoprotein gene structure and consists of four exons and three introns. Minor differences exist between the monkey and human structural gene sequence but do not result in significant changes. Sequences preceding the putative monkey apo A–I promoter are virtually identical to the human apo A–I enhancer region with an exact match to the three enhancer DNA binding sites.

The murine leukemia inhibition factor gene (Lif) is located on proximal chromosome 11, not chromosome 13.

Lif, the murine gene encoding leukemia inhibition factor (LIF), has been previously localized to proximal Chromosome (Chr) 11. Hilda, the murine gene encoding "human interleukin in DA cells" (HILDA) has been localized to Chr 13. Since these two growth factors are identical, the proposal for two different structural loci is intriguing. To address this issue, blot hybridization methods have been used to establish the position of the structural gene sequence unambiguously. DNAs from somatic cell hybrids, recombinant inbred mice, and backcross mice have been probed with a sequence that encodes LIF/HILDA. The results support the assignment of this sequence to proximal Chr 11. These studies also establish a synteny group, including Lif and Tcn-2, the structural gene for transcobalamin 2, that is conserved between man and mouse.

Transcriptional regulation of cytochrome d in nitrogen-fixing Azotobacter vinelandii. Evidence that up-regulation during N2 fixation is independent of nifA but dependent on ntrA.

Cytochrome d has been postulated to be the "respiratory protection" oxidase of Azotobacter vinelandii, allowing this organism to fix nitrogen under aerobic growth conditions. We have previously cloned and characterized the structural genes for the A. vinelandii cytochrome d (cydA and cydB). The cyd genes are co-transcribed, yielding an mRNA of approximately 3.6 kilobase pairs. The level of the cyd message was 2-3-fold higher in cells that were fixing nitrogen, as compared with non-nitrogen-fixing cells. RNase protection analysis was used to determine the transcriptional start site at 275 bases upstream of the initiator ATG of cydA, and this start site was the same for nitrogen-fixing and non-nitrogen-fixing cells. The cyd promoter has sequence similarities to the canonical Escherichia coli promoters, which are transcribed by the major sigma 70 form of RNA polymerase. Plasmid-borne lacZ transcriptional fusions were constructed, using approximately 650 base pairs of 5'-upstream sequences of the cyd structural genes. This region had a strong promoter activity which was further up-regulated 1.5-2.5-fold upon the induction of nitrogen fixation. The cyd-lacZ fusions were characterized in a nifA- as well as an ntrA- background. Mutations in neither of these nif regulatory genes affected the constitutive expression of cyd under non-nitrogen-fixing conditions. However, the up-regulation of this promoter during the induction of nitrogen fixation was abolished only in the ntrA- background. Based on these results, the cytochrome d promoter of A. vinelandii belongs to a new class of nitrogen-regulated promoters which, unlike the authentic nif genes, does not require the ntrA gene product for its expression. The up-regulation of this promoter during nitrogen fixation, however, requires the ntrA gene product.

Hyperexpression in Escherichia coli, purification, and characterization of the metallo-β-lactamase of Bacillus cereus 5/B/6

We used site-directed mutagenesis to introduce both a NdeI restriction endonuclease site and an initiator codon at the junction of the leader and structural gene sequences of the metallo-β-lactamase of Bacillus cereus 5/B6. This construct allowed us to clone just the β-lactamase structural gene sequence into an Escherichia coli expression vector. E. coli cells were transformed with the recombinant plasmid, the B. cereus β-lactamase was expressed, and these E. coli cells were disrupted by sonic oscillation. When the resultant suspensions were clarified by ultracentrifugation, the B. cereus β-lactamase represented 15% of the total protein in the supernatant. Subsequent gel filtration and ionexchange chromatography allowed the first reported purification to homogeneity of the B. cereus β-lactamase from E. coli with an 87% recovery and an overall yield of 17 mg of enzyme per liter of cell culture. The electrophoretic mobilities of the enzyme expressed in and purified from E. coli and the enzyme purified directly from B. cereus were identical in both native and sodium dodecyl sulfate gel electrophoreses. As with the B. cereus enzyme, K m and V max (using cephalosporin C as substrate) for the enzyme purified from E. coli were 0.39 mM and 1333 units/mg protein, respectively. Likewise, the Co(II)-reconstituted enzyme purified from E. coli, which retained 29% of the activity of the Zn(II) enzyme, had electronic absorption spectra with maxima at 347, 551, 617, and 646 nm with extinction coefficients of 900, 250, 173, and 150 m −1 cm −1, respectively.

Non-radioactively labelled polynucleotide and oligonucleotide DNA probes, for selectively detecting Escherichia coli strains producing Vero cytotoxins VT1, VT2 and VT2 variant

Vero cytotoxin producing Escherichia coli (VTEC) were detected in faecal specimens and bacterial isolates, using non-radioactively labelled polynucleotide and oligonucleotide DNA probes specific for Vero cytotoxin (VT) genes. VT1 and VT2 structural gene sequences, previously cloned and used for radioactive probes, were labelled with digoxigenin or biotin. Oligonucleotide gene sequences coding for the A subunit of VT1, VT2 and VT2 variant were labelled with digoxigenin. The VT1 and VT2 probes were specific for detecting VT1 and VT2 gene sequences and gave very similar results to those obtained using the radioactive label 35S as a standard. The VT2 variant probe hybridized only with the strains of porcine origin. For the range of isolates tested, there was little significant difference in specificity and sensitivity between the digoxigenin-labelled polynucleotide and oligonucleotide probes. The biotin system gave rise to more non-specific effects, particularly with some non- E. coli strains, and was therefore less reliable. All of the digoxigenin-labelled probes gave satisfactory results after several times re-use, which is of importance when considering cost.

Sequence of a sea urchin hsp70 gene and its 5′ flanking region

We report the nucleotide sequence of a 4470-bp fragment derived from a sea urchin genomic clone containing part of a heat-shock protein 70 (Hsp 70)-encoding gene. This fragment, named hsp70 gene II, contains 1271 bp of the flanking region and 3299 bp of structural gene sequence interrupted by five introns and encoding the N-terminal 371 amino acids (aa) of the protein. The 5′ flanking region contains a putative TATA element, two CCAAT boxes, four heat-shock consensus sequence elements ( hse) and one consensus sequence for binding of Sp1. Remarkable homologies were observed for deduced aa sequence and intron-exon organization between hsp70 gene II and rat hsc73 gene.

DNA sequence of the purC gene encoding 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide synthetase and organization of the dapA-purC region of Escherichia coli K-12

5'-Phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide synthetase (EC 6.3.2.6), encoded by the purC gene of Escherichia coli K-12, catalyzes the synthesis of 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide from 5'-phosphoribosyl-5-aminoimidazole-4-carboxylic acid. The mature protein, as deduced from the purC structural gene sequence, contains 237 amino acids and has a calculated Mr of 26,998. The control region of the purC gene was identified by primer extension mapping of the 5' end of the purC mRNA. The purC control region contains a binding site for and is regulated by the purine repressor, the product of the purR gene. An unusual feature of the 5' untranslated region of the purC mRNA is the presence of a repetitive extragenic palindrome sequence normally found in intercistronic or 3' untranslated regions. The DNA sequence was extended 1.281 kilobases upstream of the purC structural gene and overlapped with the previously determined dapA sequence. Termination of transcription from the dapA-purC intercistronic region may occur within the -35 region of the purC control region. The purC gene has been positioned on the E. coli restriction map and is transcribed in a counterclockwise direction.

Nucleotide sequence of the hydrogenase structural genes from Rhizobium leguminosarum

The nucleotide sequence of a DNA fragment (total lengh of 3052 nucleotides) from transcriptional unit hup1 is described. It contains the coding regions for the polypeptide subunits of the R. leguminosarum hydrogenase plus the upstream flanking region.

Identification of a bacteriophage containing a silent staphylococcal variant enterotoxin gene (sezA+)

A variant enterotoxin gene, referred to as sezA+, has been identified. Staphylococcus aureus FRI1106, a staphylococcal enterotoxin type D producer (Sed+), contained HindIII fragments of 3.8 and 9.4 kilobase pairs (kbp) that hybridized in Southern blot analysis to a probe containing only staphylococcal enterotoxin type A structural gene sequences. Presumably, probe A-624 hybridized to the 9.4-kbp HindIII fragment because of the sequence homology between sea+ and sed+. This 9.4-kbp HindIII fragment, which was part of a staphylococcal plasmid, was isolated and ligated into an Escherichia coli plasmid vector; Sed+ E. coli recombinant clones were isolated. The 3.8-kbp HindIII fragment was shown to be part of a viable lysogenic bacteriophage, and it contained sezA+. This sezA(+)-containing fragment was cloned into E. coli, and its DNA sequence was determined. Examination of the nucleotide sequence revealed a 771-bp region that contained an open reading frame with 85 and 77% nucleotide and derived amino acid sequence identifies with sea+ and staphylococcal enterotoxin type A, respectively. This open reading frame has 83 to 50% nucleotide sequence identities with the other types of staphylococcal enterotoxin genes. sezA+ was shown to be transcribed into stable mRNA. However, the sezA+ mRNA was not translated into an enterotoxinlike protein because it lacks an appropriate translation initiation codon.

Glycinamide ribonucleotide synthetase from Escherichia coli: cloning, overproduction, sequencing, isolation, and characterization

The purD gene of Escherichia coli encoding the enzyme glycinamide ribonucleotide (GAR) synthetase, which catalyzes the conversion of phosphoribosylamine (PRA), glycine, and MgATP to glycinamide ribonucleotide, MgADP, and Pi, has been cloned and sequenced. The protein, as deduced by the structural gene sequence, contains 430 amino acids and has a calculated Mr of 45,945. Construction of an overproducing strain behind a lambda pL promoter allowed a 4-fold purification of the protein to homogeneity. N-Terminal sequence analysis and comparison of the sequence with those of other GAR synthetases confirm the amino acid sequence deduced from the gene sequence. Initial velocity studies and product and dead-end inhibition studies are most consistent with a sequential ordered mechanism of substrate binding and product release in which PRA binds first followed by MgATP and then glycine; Pi leaves first, followed by loss of MgADP and finally GAR. Incubation of [18O]glycine, ATP, and PRA results in quantitative transfer of the 18O to Pi. GAR synthetase is very specific for its substrate glycine.

Cloning and Characterization of a Disease Resistance Response Gene in Pea Inducible byFusarium solani

Disease resistance response genes (DRRG) of peas are expressed as the tissue is expressing race-specific or nonhost resistance. A pea genomic clone DRRG49-c encompassing one DRRG structural gene, the expression of which is correlated with the expression of disease resistance, was sequenced and characterized. The 2.3-kb genomic segment sequenced encompassed 986 bp 5' to the major transcriptional initiation site, a 474-bp open-reading frame interrupted by one 88-bp AT-rich intron and an additional 574-bp segment 3' from the stop codon. Southern blot analysis indicated that the DRRG structural gene is one of a multigenic family, and an estimated five copies exist within the pea genome. Primer extension analysis of the 5' terminus of the corresponding RNA suggested the presence of one major transcript with possibly two minor transcripts. The major transcript, located 65 bp from the translational initiation site, was expressed when challenged with Fusarium solani f. sp. phaseoli but not with water. The structural gene sequence corresponding to the genomic clone DRRG49-c is not identical with the structural gene of the cDNA clone DRRG49-a used as a probe for northern blot analysis, and thus, a possibility remains that it is not expressed in peas; however, the DRRG49-c promoter was able to express the chloramphenicol acetyltransferase reporter gene in tobacco protoplasts. Western blot analysis using antiserum prepared from a beta-galactosidase-DRRG49-a fusion protein identified the DRRG49 gene product as a major protein accumulating during the host-pathogen interactions.