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PubMed1989

Evaluation of evolutionary divergence in the genus Naegleria by analysis of ribosomal DNA plasmid restriction patterns.

Ribosomal DNA (rDNA) plasmid restriction maps of 10 strains and rDNA hybridisation patterns of 61 additional strains have been used to assess inter- and intra-specific diversity and phylogenetic relationships in the genus Naegleria. The results obtained by this method largely confirm those of previous studies based on a variety of other criteria. They indicate that very little variation exists within the pathogenic species Naegleria fowleri despite its worldwide distribution and that it is closely related to the nonpathogenic Naegleria lovaniensis. Naegleria gruberi is most likely a polyphyletic grouping and care should be taken when using one strain as a reference point for this species. In addition, the two subspecies of the pathogenic Naegleria australiensis arose separately from within the range of variability encompassed by N. gruberi, as did the species Adelphamoeba galeacystis which should probably be assigned to the genus Naegleria. The species Naegleria jadini and Naegleria andersoni are not closely related to any other in the genus based on their rDNA patterns.

Animals↗
PubMed2005

Identification of a key amino acid residue of Streptomyces phospholipase D for thermostability by in vivo DNA shuffling.

To isolate thermostability-related amino acid residues of Streptomyces phospholipase D (PLD), we constructed a chimeral genes library between two highly homologous plds, which exhibited different thermostabilities, by an in vivo DNA shuffling method using Escherichia coli that has a mutation of a single-stranded DNA-binding protein gene. To confirm the location of the recombination site, we carried out the restriction mapping of 68 chimeral pld genes. The recombination sites were widely dispersed over the entire pld sequence. Moreover, we examined six chimeral PLDs by comparing their thermostabilities with those of parental PLDs. To identify a thermostability-related amino acid residue, we investigated the thermostability of chimera C that was the most thermolabile among the six chimeras. We identified the thermostability-related factor Gly-188, which is located in the alpha-7 helix of PLD from Streptomyces septatus TH-2 (TH-2PLD). TH-2PLD mutants, in which Gly-188 was substituted with Phe, Val or Trp, exhibited higher thermostabilities than that of the parental PLD. Gly-188 substituted with the Phe mutant, which was the most stable among the mutants, showed an enzyme activity almost the same as that of TH-2PLD as determine by kinetic analysis.

Amino Acid Sequence↗
PubMed1995

Mitochondrial DNA and Ascaris microepidemiology: the composition of parasite populations from individual hosts, families and villages.

Patterns of genetic subdivision in parasite populations can provide important insights into transmission processes and complement information obtained using traditional epidemiological techniques. We describe mitochondrial sequence variation in 265 Ascaris collected from 62 individual hosts (humans and pigs) from 35 households in 3 Guatemalan locations. Restriction mapping of individual worms revealed 42 distinct mitochondrial genotypes. We ask whether the mitochondrial genotypes found in worms from individual hosts, from families of hosts and from villages represent random samples from the total Ascaris population. Patterns of genetic subdivision were quantified using F-statistics, while deviations from the null hypothesis of randomness were evaluated by a simple resampling procedure. The analysis revealed significant deviations from panmixia. Parasite populations were strongly structured at the level of the individual host in both humans and pigs: parasites bearing the same mitochondrial genotype were found more frequently than would be expected by chance within hosts. Significant heterogeneity was also observed among populations from different villages, but not from different families within a village. The clustering of related parasites within hosts suggests a similar clustering of related infective stages in the environment and may explain why sex ratios in Ascaris are female-biased. We discuss aspects of Ascaris biology which may lead to the observed patterns.

Animals↗
PubMed1996

The repB gene required for production of extracellular enzymes and fluorescent siderophores in Pseudomonas viridiflava is an analog of the gacA gene of Pseudomonas syringae.

Two genes, designated repA and repB, are involved in the regulation of the synthesis of extracellular pectate lyase, protease, and alginate in Pseudomonas viridiflava. The repA gene has been shown to encode a protein highly homologous to several bacterial sensors in the two-component regulator family including the LemA of Pseudomonas syringae. In this study, the repB locus, initially identified in a 6.3-kb EcoRI genomic fragment of P. viridiflava, was further characterized. Results obtained from restriction mapping, deletion subclonings, and mini-Mu-LacZ fusions indicated that the repB gene was contained within a 0.8-kb HindIII-PstI region. Sequence analysis of this repB region revealed the presence of an open reading frame, which was predicted to encode a protein similar or identical to the gacA response regulator found in P. syringae and Pseudomonas fluorescens. The repB gene of P. viridiflava also regulated the production of fluorescent siderophores, in addition to the aforementioned extracellular enzymes and alginate. The repB or gacA homologs were detected in the genomes of nine other strains of P. viridiflava, P. fluorescens, and P. syringae included in the study. The data presented here and earlier indicate that the repA/repB gene regulatory system of P. viridiflava is analogous to the lemA/gacA system of P. syringae and P. fluorescens.

Alginates↗
PubMed1989

Isolation and characterization of rat cytochrome P-450IIB gene family members. Use of the polymerase chain reaction to detect expression of a novel P-450 mRNA.

The rat cytochrome P-450IIB gene family consists of at least 10 members, 2 of which, P-450b and P-450e, have been well characterized and are activated transcriptionally by phenobarbital (PB) in the liver. The remaining genes in this family have not been studied extensively. In this report, data are presented that provide additional characterization of a P-450IIB gene, termed gene IV. The complete gene IV and flanking regions were isolated from a rat liver Charon 4A genomic library and subjected to a variety of analyses. Structural homology to the P-450b gene was confirmed by comparative restriction mapping and high stringency hybridization of gene IV fragments to probes comprising the entire cDNA for P-450b. The 5'-portion of gene IV, including its flanking region, was sequenced and contained an open reading frame for 58 amino acids which were 62% related to exon 1 of the P-450b/e genes. Typical TATA and CAAT promoter elements, as well as two Sp1 core sequences and a site related to a glucocorticoid responsive element were found in gene IV. Northern blot studies with an oligomer probe specific to gene IV sequence indicated a 4.3-kilobase pair polyadenylated transcript present at low levels in untreated rat liver and inducible approximately 6-fold by PB treatment. Primer extension experiments indicated that the transcription initiation site mapped to a position on gene IV that was analogous to that reported for the structurally similar P-450e gene. Due to the low levels of hepatic RNA expression, we employed the polymerase chain reaction to facilitate characterization of gene IV transcripts. The polymerase chain reaction data verified that gene IV transcripts were elevated after PB treatment. Significantly, the polymerase chain reaction results demonstrated that gene IV transcripts were associated with hepatic polysome fractions, indicating their active utilization in this tissue. Polymerase chain reaction analysis of RNA also indicated constitutive expression of gene IV transcripts in rat lung, kidney, and testis, as well as fetal rat liver. Together, these data show that gene IV is a transcribed member of the P-450IIB gene family and, like the well characterized P-450b and P-450e genes, is positively regulated by PB in rat liver.

Animals↗
PubMed1993

Chromosome t(11;14)(q13;q32) breakpoints in centrocytic lymphoma are highly localized at the bcl-1 major translocation cluster.

Chromosome 11q13 translocation breakpoints have been found dispersed over more than 100 kb of genomic DNA in centrocytic lymphoma and lymphocytic lymphoma of intermediate differentiation (mantle cell lymphoma). Approximately one-half of these translocations occur at the bcl-1 major translocation cluster (MTC) and appear tightly clustered by Southern blot restriction mapping. In order to specifically characterize these t(11;14)(q13;q32) translocations, six cases of centrocytic lymphoma with MTC rearrangements on Southern blot were studied. Genomic DNA was amplified by PCR (polymerase chain reaction) using a consensus immunoglobulin heavy-chain joining gene (JH) primer and two separate MTC primers. Sequencing of the PCR products revealed the MTC breakpoints in all six cases to be clustered within a 61 basepair span, very similar to those previously reported in t(11;14)-containing human B-cell lines. No two breakpoints were identical. JH breakpoints involved JH4 in five cases and JH6 in the other. Tight clustering of the MTC breakpoints thus permits PCR detection of the t(11;14) in many cases of centrocytic lymphoma, which may be of value in classifying this subtype of non-Hodgkin's lymphoma.

Base Sequence↗
PubMed2003

Detailed characterization of a homozygously deleted region corresponding to a candidate tumor suppressor locus at distal 17p13.3 in human lung cancer.

17p13.3 is one of the chromosomal regions most frequently affected by allelic loss in a variety of human neoplasms including lung cancer. A number of loss of heterozygosity (LOH) analyses have suggested the existence of a tumor suppressor gene at 17p13.3, distal to the p53 locus at 17p13.1. In the present study, we characterized a homozygous deletion at 17p13.3 in a small cell lung cancer cell line by constructing a bacterial artificial chromosome (BAC) contig and a restriction map surrounding the region, as well as by utilizing publicly available draft sequences. We defined the breakpoint, assigned and analysed two known genes, 14-3-3 epsilon and CRK, and a novel gene LOST1 within or at the end of the homozygous deletion of about 170 kb in size. Marked reduction of LOST1 expression was detected in 69% (11/16) of lung cancer specimens by quantitative real-time RT-PCR, while significant DNA hypermethylation was observed at the 5' end of the LOST1 gene in four of six lung cancer cell lines with negligible LOST1 expression. We also show here that a polymorphic marker D17S1174, which resides within the homozygous deletion, was apparently located in the middle of the minimum LOH region, providing further supportive evidence for the presence of a tumor suppressor gene(s) in this region.

Amino Acid Sequence↗
PubMed1996

Cloning of the genes for and characterization of the early stages of toluene and o-xylene catabolism in Pseudomonas stutzeri OX1.

In order to study the toluene and o-xylene catabolic genes of Pseudomonas stutzeri OX1, a genomic library was constructed. A 28-kb EcoRI restriction endonuclease DNA fragment, cloned into the vector plasmid pLAFR1 and designated pFB3401, permitted Pseudomonas putida PaW340 to convert toluene and o-xylene into the corresponding meta-ring fission products. Physical and functional endonuclease restriction maps have been derived from the cloned DNA fragment. Further subcloning into and deletion analysis in the Escherichia coli vector pGEM-3Z allowed the genes for the conversion of toluene or o-xylene into the corresponding catechols to be mapped within a 6-kb region of the pFB3401 insert and their direction of transcription to be determined. Following exposure to toluene, E. coli cells carrying this 6-kb region produce a mixture of o-cresol, m-cresol, and p-cresol, which are further converted to 3-methylcatechol and 4-methylcatechol. Similarly, a mixture of 2,3-dimethylphenol and 3,4-dimethylphenol, further converted into dimethylcatechols, was detected after exposure to o-xylene. The enzyme involved in the first step of toluene and o-xylene degradation exhibited a broad substrate specificity, being able to oxidize also benzene, ethylbenzene, m-xylene, p-xylene, styrene, and naphthalene. Deletions of the 6-kb region which affect the ability to convert toluene or o-xylene into the corresponding methylphenols compromise also their further oxidation to methylcatechols. This suggests that a single enzyme system could be involved in both steps of the early stages of toluene and o-xylene catabolism.

Benzene↗
PubMed1999

The chlorocatechol-catabolic transposon Tn5707 of Alcaligenes eutrophus NH9, carrying a gene cluster highly homologous to that in the 1,2,4-trichlorobenzene-degrading bacterium Pseudomonas sp. strain P51, confers the ability to grow on 3-chlorobenzoate.

Alcaligenes eutrophus (Ralstonia eutropha) NH9, isolated in Japan, utilizes 3-chlorobenzoate as its sole source of carbon and energy. Sequencing of the relevant region of plasmid pENH91 from strain NH9 revealed that the genes for the catabolic enzymes were homologous to the genes of the modified ortho-cleavage pathway. The genes from strain NH9 (cbnR-ABCD) showed the highest homology (89 to 100% identity at the nucleotide level) to the tcbR-CDEF genes on plasmid pP51 of the 1,2,4-trichlorobenzene-degrading bacterium Pseudomonas sp. strain P51, which was isolated in The Netherlands. The structure of the operon, including the lengths of open reading frames and intervening sequences, was completely conserved between the cbn and tcb genes. Most nucleotide substitutions were localized within and proximal to the cbnB (tcbD) gene. The difference in the chloroaromatics that the two strains could use as growth substrates seemed to be due to differences in enzymes that convert substrates to chlorocatechols. The restriction map of plasmid pENH91 was clearly different from that of pP51 except in the regions that contained the cbnR-ABCD and tcbR-CDEF genes, respectively, suggesting that the chlorocatechol gene clusters might have been transferred as units. Two homologous sequences, present as direct repeats in both flanking regions of the cbnR-ABCD genes on pENH91, were found to be identical insertion sequences (ISs), designated IS1600, which formed a composite transposon designated Tn5707. Although the tcbR-CDEF genes were not associated with similar ISs, a DNA fragment homologous to IS1600 was cloned from the chromosome of strain P51. The sequence of the fragment suggested that it might be a remnant of an IS. The two sequences, together with IS1326 and nmoT, formed a distinct cluster on a phylogenetic tree of the IS21 family. The diversity of the sources of these IS or IS-like elements suggests the prevalence of ISs of this type.

Amino Acid Sequence↗
PubMed2000

In vitro DNA fragmentation of mitochondrial DNA caused by single-stranded breakage related to macroplasmodial senescence of the true slime mold, Physarum polycephalum.

We found that mitochondrial DNA (mtDNA) isolated from Physarum polycephalum fragmented itself in weak ionic solutions. The mtDNA was dissolved in STE (saline Tris-EDTA: 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA), TE (10 mM Tris-HCl, 1 mM EDTA) and DW, and then electrophoresed in an agarose gel. The intact 86-kbp mtDNA band was seen in STE, but several novel bands appeared in TE and DW. In TE, two discrete bands appeared at 6.7-kbp (alpha-band) and 5.0-kbp (beta-band), whereas at least 17 discrete bands were observed in distilled water (DW). These fragmentation patterns were not stoichiometric, as seen when using restriction endonucleases, but were clearly different from the degradation of DNA caused by a physical shearing force or a contaminating nuclease. In this paper, we characterize this in vitro fragmentation of mtDNA from P. polycephalum. We located 19 fragments, including the alpha and beta fragments, on a mtDNA restriction map, and demonstrated that these cleavage sites were S1 nuclease-sensitive regions, which are single-stranded DNA regions such as nicks and gaps in the mtDNA. The alpha and beta fragments are derived from the region encoding ribosomal RNAs (rRNAs) and the ATP synthase (atpA) gene, while the other 17 fragments are not derived from any specific region, but the cleavage sites are located throughout the mtDNA molecule. In P. polycephalum, it is well known that the growth rate of macroplasmodia decreases with aging. Equal amounts of mtDNA from juvenile and aged macroplasmodia were electrophoresed and the frequency of the beta fragment in each sample was measured. The ratio of the beta band to the total signal including background was estimated to be 3.3-4.0% in juvenile macroplasmodia, whereas it increased to 8.3-28.2% in aged macroplasmodia. This result suggests that the in vitro fragmentation of mtDNA is associated with macroplasmodial senescence. The single-stranded breakage of mtDNA of P. polycephalum may accumulate with age.

Aging↗
PubMed1992

Characterization of a low copy repetitive element S232 involved in the generation of frequent deletions of the distal short arm of the human X chromosome.

There are several copies of related sequences on the distal short arm of the human X chromosome and the proximal long arm of the Y chromosome which were originally detected by cross hybridization with a genomic DNA clone, CRI-S232. Recombination between two S232-like sequences flanking the steroid sulfatase locus has been shown to cause frequent deletions in the X chromosome short arm, resulting in steroid sulfatase deficiency. We now report the characterization of several S232-like sequences. Restriction mapping and sequence analysis show that each S232 unit contains 5 kb of unique sequence in addition to two elements, RU1 and RU2, composed of a variable number of tandem repeats. RU1 consists of 30 bp repeating units and its length shows minimal variation between individuals. The RU2 elements in the hypervariable S232 loci on the X chromosome consist of repeating sequences which are highly asymmetric, with about 90% purines and no C's on one strand. The X-derived RU2 elements range from 0.6 kb to over 23 kb among different individuals, accounting entirely for the observed polymorphism at the S232 loci. Although the repeating units of the RU2 elements in the nonpolymorphic S232 loci on the Y chromosome share high sequence homology with those on the X chromosome, they exhibit much higher intrarepeat sequence variation. S232 homologous sequences are found in great apes, old world and new world monkeys. In chimpanzees and gorillas the S232-like sequences are polymorphic in length.

Animals↗
PubMed1996

Construction and use of recombinant Escherichia coli strains for the synthesis of toluene cis-glycol.

The toluene dioxygenase genes derived from Pseudomonas putida NCIMB 11767 were subcloned from a previously constructed recombinant plasmid, pIG, using pUC18 as the cloning vector and E. coli TG2 as the host strain. The resulting strain, E. coli TG2 (p1/1), produced toluene cis-glycol when grown in LB broth or minimal medium in the presence of toluene. Restriction mapping and partial DNA sequencing provided evidence for the presence of ORFs with extensive homology to parts of the tod operon from P. putida F1. The clones exhibited some residual toluene cis-glycol dehydrogenase activity which resulted in the formation of small amounts of 3-methylcatechol. Expression of the dioxygenase was induced by toluene, but was not directed by the lac promoter within the cloning vector. The clones were assessed for toluene cis-glycol production in pH-controlled batch cultures, and the maximum product concentration obtained was 1.02 g l-1. Product formation was dependent upon the presence of glucose in the culture medium. Although the substrate was toxic, the biotransformation was apparently limited by the supply of toluene. The results suggest that it should be possible to improve toluene cis-glycol production by recombinants substantially by improving both the strain and fermentation process.

Amino Acid Sequence↗
PubMed1993

Genetic structure and epidemiology of Ascaris populations: patterns of host affiliation in Guatemala.

In Guatemalan villages people commonly rear pigs, and both hosts may be infected with Ascaris. This study was designed to ask whether both humans and pigs are potential hosts in a single parasite transmission cycle in such villages, or alternatively, if there are two separate transmission cycles, one involving pigs and one involving human hosts. Parasites were collected from both host species from locations in the north and south of Guatemala. Allelic variation in the nuclear genome of Ascaris was measured using enzyme electrophoresis, while mitochondrial DNA (mtDNA) sequence variation was quantified using restriction mapping. Low levels of enzyme polymorphism were found in Ascaris, but allele frequencies at two loci, mannose phosphate isomerase and esterase, suggest that there is little gene exchange between parasite populations from humans and pigs. MtDNA haplotypes fall into two distinct clusters which differ in sequence by 3-4%; the two clusters broadly correspond to worms collected from humans and those collected from pigs. However, some parasites collected from humans have mtDNA characteristic of the 'pig Ascaris' haplotype cluster, while some parasites collected from pigs have mtDNA characteristic of the 'human Ascaris' haplotype cluster. These shared haplotypes are unlikely to represent contemporary cross-infection events. Patterns of phylogenetic similarity and geographical distribution of these haplotypes suggest, instead, that they are the result of two historical introgressions of mtDNA between the two host-associated Ascaris populations. The results clearly demonstrate that Ascaris from humans and pigs are involved in separate transmission cycles in Guatemala.

Alleles↗
PubMed1992

Cytoplasmic male sterility is associated with large deletions in the mitochondrial DNA of two Nicotiana sylvestris protoclones.

Two cytoplasmic male-sterile plants (CMSI and CMSII) were obtained by protoplast culture in Nicotiana sylvestris. Both plants showed large deletions (up to 50 kb) in their mitochondrial DNA. Restriction maps of the reorganized regions suggested that the deletions occurred via two homologous recombination events (rec. 1 and rec. 2) in the parental mitochondrial genome. With the exception of nad5, no mitochondrial DNA polymorphism could be detected between parental and CMS lines using different heterologous genes probes. A sequence homologous to the Oenothera nad5 mitochondrial gene was located close to the CMSI-specific rec. 2 region. Moreover, a cDNA probe corresponding to total mitochondrial RNA from the parent line was found to hybridize to mitochondrial DNA fragments involved in the rec. 1 event common to both CMS lines, suggesting that rec. 1 lies in a transcribed region. Cytoplasmic male sterility in the Nicotiana sylvestris CMS mutants could be due either to gene deletion or to a regulatory effect of such a deletion on mitochondrial gene expression, rather than to the presence of specific polypeptides as has been shown in the T cytoplasm of maize, or in CMS Petunia.

Blotting, Southern↗
PubMed1993

Type 2X-myosin heavy chain is coded by a muscle fiber type-specific and developmentally regulated gene.

We have previously reported the identification of a distinct myosin heavy chain (MyHC) isoform in a major subpopulation of rat skeletal muscle fibers, referred to as 2X fibers (Schiaffino, S., L. Gorza, S. Sartore, L. Saggin, M. Vianello, K. Gundersen, and T. Lømo. 1989. J. Muscle Res. Cell Motil. 10:197-205). However, it was not known whether 2X-MyHC is the product of posttranslational modification of other MyHCs or is coded by a distinct mRNA. We report here the isolation and characterization of cDNAs coding a MyHC isoform that is expressed in type 2X skeletal muscle fibers. 2X-MyHC transcripts differ from other MyHC transcripts in their restriction map and 3' end sequence and are thus derived from a distinct gene. In situ hybridization analyses show that 2X-MyHC transcripts are expressed at high levels in the diaphragm and fast hindlimb muscles and can be coexpressed either with 2B- or 2A-MyHC transcripts in a number of fibers. At the single fiber level the distribution of each MyHC mRNA closely matches that of the corresponding protein, determined by specific antibodies on serial sections. In hindlimb muscles 2X-, 2A-, and 2B-MyHC transcripts are first detected by postnatal day 2-5 and display from the earliest stages a distinct pattern of distribution in different muscles and different fibers. The emergence of type 2 MyHC isoforms thus defines a distinct neonatal phase of fiber type differentiation during muscle development. The functional significance of MyHC isoforms is discussed with particular reference to the velocity of shortening of skeletal muscle fibers.

Amino Acid Sequence↗
PubMed1991

Extended MHC haplotypes and CYP21/C4 gene organisation in Irish 21-hydroxylase deficiency families.

We have analysed fifteen classical 21-hydroxylase deficiency families from throughout Southern Ireland and report the serologically defined HLA-A, HLA-B, HLA-Cw, HLA-DR, C4A and C4B polymorphisms that characterize the inferred disease haplotypes. Additionally, we have used a combination of short and long range restriction mapping procedures in order to characterize the CYP21/C4 gene organization associated with individual serologically defined haplotypes. The results obtained indicate that disease haplotypes are characterized by a high frequency (33%) of CYP21B gene deletion and 8 out of 10 such deletion haplotypes are represented by the extended haplotype HLA-DR1, C4BQo, C4A3, HLA-B40(w60), HLA-Cw3, HLA-A3. Large scale length polymorphism in the CYP21/C4 gene cluster was found to conform strictly to a variable number of tandem repeats model with 4 alleles being detected. Disease haplotypes in which defective CYP21B gene expression is inferred to result from pathological point mutations show extensive diversity of associated HLA markers and include two examples of the extended HLA haplotype HLA-DR3, B8, Cw7, A1 haplotype, which has previously been reported to be negatively associated with 21-hydroxylase deficiency. One unusual disease haplotype has two CYP21 + C4 units, both of which appear to contain CYP21B-like genes.

Adrenal Hyperplasia, Congenital↗
PubMed1993

Cloning and genetic analysis of the Vibrio vulnificus fur gene and construction of a fur mutant by in vivo marker exchange.

Vibrio vulnificus infections have been associated with iron overload and preexisting liver disease. Iron may play a major role in the pathogenesis of V. vulnificus infections. Many virulence genes, as well as genes involved in the transport of iron by bacteria, are regulated by iron, with increased expression under low-iron conditions. In Escherichia coli and Vibrio cholerae, transcriptional regulation by iron depends on the fur gene. We utilized Southern hybridization under low- and high-stringency conditions with both E. coli and V. cholerae fur gene probes to demonstrate that there are fur-homologous sequences in the DNAs of V. vulnificus, Vibrio fischeri, and Aeromonas sp. but not in the DNAs of the other bacterial species tested. We developed a restriction map and cloned the fur-homologous sequence from V. vulnificus. The hybridizing clone of V. vulnificus chromosomal DNA complemented a V. cholerae fur mutant. DNA sequence analysis confirmed the presence of a 149-amino-acid open reading frame that was 77% homologous to E. coli Fur and 93% homologous to V. cholerae Fur. Primer extension localized a single promoter for the V. vulnificus fur gene. Northern (RNA) blot analysis and beta-galactosidase assays of an operon fusion to lacZ suggested that there was not significant regulation of transcription of V. vulnificus fur by iron or the E. coli Fur protein. We used marker exchange to construct a V. vulnificus fur deletion mutant and confirmed its phenotype by observing overexpression of iron-regulated outer membrane proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fur deletion mutant of V. vulnificus will be helpful in future studies of the role of iron in V. vulnificus pathogenesis.

Amino Acid Sequence↗
PubMed1992

Structure and expression of class II alpha genes in miniature swine.

Two overlapping genomic clones corresponding to the swine DRA class II gene were isolated and characterized. Restriction mapping and partial sequence data of the exon-containing fragments allowed identification and orientation of the five exons encoding the alpha chain. Two full length cDNA clones corresponding to the transcribed DRA gene from two different haplotypes of the swine MHC were sequenced. Nucleotide sequence alignments revealed that the two swine DRA cDNA were very similar and closely related to the human DRA equivalent. An additional glycosylation site, compared with those of human DRA, was found in the second external domain of the protein. Northern analyses showed that porcine DRA and DQA genes were the only two class II alpha genes expressed in the spleen, despite the presence of DPA and DZA genes in the genome. In addition to transfected cells expressing homologous pairs of alpha and beta chains from SLA-DR, stable transfectants expressing nonhomologous pairs of alpha and beta chains from DR and DQ loci were obtained, suggesting that such associations may contribute to the functional heterogeneity of class II products.

Animals↗

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