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PubMed1994

Transcribed repetitive DNA sequences in telomeric regions of rice (Oryza sativa).

We have isolated and characterized from rice three repetitive DNA sequences, Os48, Osc-567, and OsG3-430. Our results indicate that these repetitive sequences are highly transcribed, and transcripts complementary to both strands of the Os48 family of sequences account for up to 3% of the total cellular RNA. Pulsed-field gel electrophoresis, restriction mapping, and DNA sequence analyses have revealed a complex pattern of structural organization of the three families of repetitive sequences. Os48 and Osc-567 are organized in long tandem arrays, whereas copies of the OsG3-340 sequence are interspersed with other sequences including arrays of the Os48 and Osc567 families. Interestingly, the three families of repetitive sequences are closely linked not only to each other, but also to telomeric sequences of rice, suggesting that transcription of these repetitive sequences may occur in regions very close to telomeres in rice.

Base Sequence↗
PubMed1991

Characterization of circular mitochondrial plasmids in three Pythium species.

Four circular plasmids, with a monomer size ranging from 3.2 to 4.94 kb, have been identified in isolates of P. aphanidermatum (two different plasmids), P. torulosum, and an unidentified echinulate isolate. The mitochondrial location has been confirmed for three of the plasmids. Each fungal isolate contained a single plasmid, present in both monomeric and oligomeric forms; plasmid monomers were present as open circles and as supercoiled forms. Restriction maps of the plasmids were dissimilar. Hybridization studies using cloned plasmids revealed no DNA sequence similarity among the different plasmids or between the plasmids and the nuclear or mitochondrial genome of the isolates from which they were recovered. Hybridization of labeled plasmid DNA to Northern transfers of mitochondrial RNA for two isolates indicate that what appears to be the predominant RNA transcript is unit length in size. For three isolates, the plasmid was retained following subculturing and was present in all asexual and sexual single-spore progeny evaluated. For one isolate of P. aphanidermatum the plasmid was unstable and was lost during subculturing.

Blotting, Northern↗
PubMed1988

Cloning of two chloramphenicol acetyltransferase genes from Clostridium butyricum and their expression in Escherichia coli and Bacillus subtilis.

Two non-homologous chloramphenicol (Cm) acetyltransferase (CAT) genes, designated catA and catB, were cloned from Clostridium butyricum type strains and characterized by restriction mapping. Both genes are efficiently expressed in Escherichia coli and Bacillus subtilis. In contrast to analogous genes from staphylococci and bacilli, gene expression is not dependent on induction by Cm. The genes are considered as chromosomal, since no association with endogenous plasmids was detectable. Southern hybridization revealed a homology between catA and the staphylococcal Cm resistance plasmid, pC194. The subunit size of the clostridial CAT enzymes expressed in E. coli was determined as 22.5 kDa (catA) and 24 kDa (catB), respectively. The C. butyricum cat genes provide potentially useful selection markers for the construction of cloning vectors from cryptic clostridial plasmids.

Bacillus subtilis↗
PubMed1989

Two regulatory genes of the maize anthocyanin pathway are homologous: isolation of B utilizing R genomic sequences.

Genetic studies in maize have identified several regulatory genes that control the tissue-specific synthesis of the purple anthocyanin pigments during development. Two such genes, R and B, exhibit extensive allelic diversity with respect to the tissue specificity and developmental timing of anthocyanin synthesis. Previous genetic studies demonstrated that certain B alleles can substitute for R function, and in these cases only one functional allele at either locus is required for pigment synthesis in the aleurone. In addition, biochemical studies have shown that both genes act on the same biosynthetic pathway, suggesting that the genes are functionally duplicate. In this report we describe DNA hybridization experiments that demonstrate that the functionally duplicate nature of B and R is reflected in DNA sequence similarity between the two genes. We took advantage of this homology and used the R genomic sequences to clone B. Two different strategies were pursued and two genomic clones isolated, a 2.5-kilobase BgIII fragment linked to the b allele in W23 inbred stocks and a 1.0-kilobase HindIII fragment linked to the B allele in CM37 stocks. Examination of several independent transposable element insertion mutations in B and revertant derivatives demonstrated that our clones recognize the functional B gene. Genomic clones representing the entire B-Peru allele were isolated, and a detailed restriction map was prepared. Using these clones we have identified a 2.2-kilobase mRNA in husks from plants containing either B-I or B-Peru alleles, but no B mRNA was detected in plants containing a b allele. The transcript is at least 100 times more abundant in strongly pigmented B-I husks than in weakly pigmented B-Peru husk tissue. Expression of functional B alleles in husk tissue correlates with the coordinate increase in mRNA levels of two structural genes of the pathway, A1 and Bz1, consistent with the postulated role of B as a regulatory gene.

Alleles↗
PubMed1989

Two cDNAs encode two nearly identical Cu/Zn superoxide dismutase proteins in maize.

SOD-4, a cytosolic form of superoxide dismutase in maize, originally was defined as a single band of activity by zymogram analysis. The protein was purified to "homogeneity" as shown by a single band on native or denaturing polyacrylamide gels and a single spot on two dimensional gels. The N-terminal amino acid sequence for the first 20 residues was determined for the purified SOD-4 protein. All residues were clearly determined except for residue twelve, where both glutamic and aspartic acids were found. A maize lambda gt11 cDNA library was constructed from scutellar poly(A)+ RNA. Two cDNAs were isolated, restriction mapped, and their DNA sequences determined. The amino acid sequence deduced from both cDNAs matched perfectly the N-terminal sequence of the purified protein except for the residue at position 12. Significantly, at the twelfth codon, one cDNA was found to code for glutamic acid and the other cDNA had a codon for aspartic acid. Both cDNAs contained similar but not identical 5' and 3' untranslated sequences. Both cDNAs contained polyadenylation signals and tails. cDNA isolations, RNA, and genomic DNA blots confirm the existence and expression of two genes that produce indistinguishable SOD-4 proteins.

Amino Acid Sequence↗
PubMed1992

The gene for autosomal dominant polycystic kidney disease lies in a 750-kb CpG-rich region.

PKD1, the locus most commonly affected by mutations that produce autosomal dominant polycystic kidney disease (ADPKD), has previously been localized to chromosome 16p13.3. Since no cytogenetic abnormalities have been found in association with ADPKD, flanking genetic markers have been required to define an interval--the PKD1 region--that contains the PKD1 gene. In this report we demonstrate, through the construction of a long-range restriction map that links the flanking genetic markers GGG1 (D16S84) and 26.6PROX (D16S125), that the PKD1 gene lies within an extremely CpG-rich 750-kb segment of chromosome 16p13.3. Approximately 90% of this region has been cloned in three extensive cosmid/bacteriophage contigs. The cloned DNA is a valuable resource for identifying new closer flanking genetic markers and for isolating candidate genes from the region.

Chromosomes, Human, Pair 16↗
PubMed1994

Isolation and in vitro expansion of lymphocytes infiltrating non-small cell lung carcinoma: functional and molecular characterisation for their use in adoptive immunotherapy.

Tumour infiltrating lymphocytes (TIL) have the capability of recognising and lysing autologous cancer cells, both in vitro and in vivo. Advanced non-small cell lung carcinoma (NSCLC) is partially insensitive to chemo radiotherapy and has a poor prognosis: thus, for this, an immunotherapeutic approach could be attempted. We expanded in vitro 46 out of 70 samples of TIL derived from NSCLC. From proliferating TILS, a number varying from 10 to 50 x 10(9) cells was obtained. These lymphocytes belonged to the T cell lineage, had the capability of growing for 45-60 days and lysed autologous better than allogeneic cancer cells. In addition, analysis of the restriction maps of T cell receptor (TRC)-beta, demonstrated that an oligoclonal population of T cells was preselected in vivo, near the tumour site, and might be expanded in vivo, using phytohaemagglutin and interleukin 2 while maintaining the same characteristics of the original population. These results give a clear rationale for the use of in vitro expanded TIL from NSCLC in protocols of adoptive immunotherapy in patients with residual disease following surgery.

Adult↗
PubMed1993

Characterization of hemolytic bacteria in subgingival plaque.

Three-quarters of the patients with periodontal diseases surveyed in this study had one or more distinct types of hemolytic bacteria in their subgingival plaque. Twelve different species of bacteria were identified, belonging to five genera (Actinomyces, Streptococcus, Staphylococcus, Prevotella, and Actinobacillus). Nine hemolytic isolates, consisting of four Prevotella denticola strains, two Actinomyces naeslundii genospecies 2 strains, and one each of P. melaninogenica, Streptococcus constellatus, and A. naeslundii genospecies 1 strains were characterized. Incorporation of pronase into blood agar medium inhibited hemolysis by all of the isolates, suggesting a proteinaceous component for each of their hemolysins. With one exception, hemolysin production appeared to be regulated by the concentration of environmental iron: exogenous hemin was found to inhibit hemolysin production, and the iron scavenging compound, 2,2'- dipyridyl, was found to promote hemolysin production by all of the strains except for the S. constellatus isolate. Genomic libraries of each of the hemolytic plaque isolates were prepared in Escherichia coli using pBR322. Hemolytic clones were isolated on blood agar medium containing ampicillin at frequencies ranging from 1-6.7 x 10(-4). Extensive restriction mapping revealed regions of homology in the case of clones derived from three P. denticola strains isolated from the same subjects. Two of the P. denticola-derived clones were virtually identical throughout the entrety of their > 5 Kb inserts. The clone derived from the third strain showed good homology to the other two within a 1.3 Kb region, but the flanking DNA showed no homology even though all three P. denticola isolates were shown to be clonally related by ribotyping.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinomyces↗
PubMed1990

Characterization of Propionibacterium plasmids.

Plasmid DNAs from 15 Propionibacterium strains were characterized by using restriction endonuclease analyses, DNA-DNA hybridizations, and curing experiments. Restriction endonuclease analysis identified seven distinct plasmids (pRGO1 through pRGO7). Detailed restriction maps were constructed for four of these plasmids. DNA-DNA hybridization analysis revealed that plasmids pRGO1 and pRGO2 had extensive sequence homology and that both were homologous to pRGO7 and to similar sequences of pRGO5. Plasmids pRGO4 and pRGO6 did not have any significant sequence homology with any of the other plasmids. Plasmid pRGO3 had partial sequence homology only with pRGO7. Curing of plasmids pRGO1, pRGO2, and pRGO5 was achieved by treatment with acriflavin, but we failed to identify any plasmid-encoded bacteriocin production, carbohydrate fermentation, or antibiotic resistance. However, physical evidence was obtained that tentatively linked the clumping phenotype of Propionibacterium jensenii P38 with plasmid pRGO5.

Base Sequence↗
PubMed1989

[The Bos taurus casein genes. Isolation and characterization of the kappa-casein gene].

A region spanning 25 kb of genomic DNA containing the kappa-casein gene, has been isolated from two genomic libraries in EMBL3 and EMBL4 phage vectors. Five phage clones containing kappa-casein gene have been found. Gene organisation has been determined using restriction mapping and a partial sequencing the 5' and 3' flanking regions. The kappa-casein gene includes 5 exons, the first of them coding for 64 nucleotides from the 5' untranslated mRNA zone. The gene is 12.5 kb long, which is almost 16 times longer than the corresponding mRNA. The first intron spans 2.5 kb, the second is the largest one and spans 5.5 kb. The 5' flanking region sequence has been analysed; it contains a TATA box from -30 to -25 bp, somewhat different from the canonic sequence, and a CAAT box at -80 bp.

Animals↗
PubMed1988

Comparison of the meta pathway operons on NAH plasmid pWW60-22 and TOL plasmid pWW53-4 and its evolutionary significance.

The regulated meta pathway operon for the catabolism of salicylate on the naphthalene plasmid pWW60-22 was cloned into the broad-host-range vector pKT230 on a 17.5 kbp BamHI fragment. The recombinant plasmid conferred the ability to grow on salicylate when mobilized into plasmid-free Pseudomonas putida PaW130. A detailed restriction map of the insert was derived and the locations of some of the genes were determined by subcloning and assaying for their gene products in Escherichia coli and P. putida hosts. The existence of a regulatory gene was demonstrated by the induction of enzyme activities in the presence of salicylate. DNA-DNA hybridization indicated a high degree of structural homology between the pWW60-22 operon and the analogous meta pathway operon on TOL plasmid pWW53-4. The data are consistent with the structural genes being arranged in an identical linear array and suggest an evolutionary link between the two catabolic systems.

Biological Evolution↗
PubMed1995

Mobile genetic elements of Fusobacterium nucleatum.

The gram-negative anaerobic bacterium, Fusobacterium nucleatum, is a predominant member of the human oral flora. As a major component of subgingival plaque, this bacterium has a significant impact on the ecology of the oral cavity due to its ability to adhere to many different microbial species. The objective of this study was to identify and characterize plasmids and transposons that may have the potential to be developed into tools for cloning, genetic transformation, and mutagenesis of oral isolates of F. nucleatum. Analysis of a collection of laboratory strains resulted in the identification of a homologous family of small cryptic plasmids. Plasmids within this family ranged in size from 6.0 to 6.6 kb. Eighteen percent of all strains examined (n = 74) contained DNA sequences related to the plasmids. Homologous plasmid sequences were found in strains belonging to 2 of the 3 subspecies of the bacterium. The 2 smallest plasmid species were cloned in Escherichia coli to facilitate endonuclease restriction mapping. Among the strains examined for plasmids, 5 exhibited resistance to at least 10 micrograms/ml of tetracycline. These strains, all members of the subsp. polymorphum, contained a tetracycline resistance determinant (TetM) as part of a Tn916-like integrated transposon sequence. The Tn916-like element and 1 of the plasmid species co-resided in a single strain of the bacterium. Hybridization patterns of the Tn916-like sequences were identical in all 5 tetracycline-resistant strains. However, these strains appeared to be clonally distinct based on genomic fingerprinting.

Base Sequence↗
PubMed1989

Physical and biological characterization of linear DNA plasmids of the yeast Pichia inositovora.

Three cryptic DNA plasmids have been identified in a strain of the yeast Pichia inositovora that are 18, 13, and 10 kbp in size. All are sensitive to digestion by DNase I, restriction endonucleases, and exonuclease III, but are resistant to the activities of RNase A and lambda exonuclease. These results indicate that each plasmid is a linear DNA molecule whose 5' ends are protected. A restriction map has been developed for each of the plasmids, demonstrating that each is unique and confirming their linear nature. The plasmids are a major constituent of DNA prepared from whole cells, but are absent from DNA preparations of purified mitochondria and nuclei, indicating that the plasmids are located in the cytoplasm. These plasmids share many of the physical characteristics described for the linear plasmids of the yeasts Kluyveromyces lactis and Saccharomycopsis crataegensis. Unlike the linear plasmids of K. lactis, however, they appear not to be capable of killer toxin production.

Autoradiography↗
PubMed1992

Distribution of interspersed repeats (Alu and Kpn) on NotI restriction fragments of human chromosome 21.

Interspersed repeated sequences (Alu and Kpn) were used as probes to detect a set of Not I restriction fragments of human chromosome 21 from the hybrid cell line WAV17. Forty different Not I fragments, ranging in size from less than 0.05 megabase (Mb) to 7.0 Mb, were identified. The total length of these fragments was 47.3 Mb. This length provides an estimate of the minimum size of the chromosome and a minimum number of fragments to be ordered to create a complete restriction map. The average length Not I fragment is 1.2 Mb. Alu and Kpn fragments are not always coincident: a 2.9-Mb fragment is detected with Kpn but not with Alu, and 13 fragments, ranging from less than 0.05 Mb to 5.6 Mb, are detected with Alu but not with Kpn; the 26 remaining fragments, covering 75% (35.3 Mb) of the total length, are detected with both repetitive probes. The presence of so many noncoincident fragments and the high variation of the hybridization signal intensities of the fragments suggest a very nonuniform distribution of Kpn and Alu repeats.

Blotting, Southern↗
PubMed1998

Taura syndrome of penaeid shrimp: cloning of viral genome fragments and development of specific gene probes.

The ssRNA genome extracted from purified Taura Syndrome Virus (TSV) was transcribed into double-stranded, blunt-ended cDNA and was used to construct cDNA libraries either in pUC 18 or in pBluescript II KS-vectors. Twelve recombinant plasmids chosen after screening of the libraries were subjected to restriction enzyme digestions for determination of size inserts and restriction maps. Two of them, pP15 and pQ1, were selected for probe construction. The inserts, 1500 and 1300 base pairs (bp) respectively, were DIG-11dUTP-labelled and the corresponding probes were named P15 and Q1. On northern blots and dot blots, using different denaturation methods, the 2 probes hybridized specifically with extracted RNA-TSV genome, TSV and infected TS shrimp homogenates. No positive hybridization was obtained with other shrimp viruses tested [Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and Hepatopancreatic Parvovirus (HPV)]. The specificity of the 2 probes was confirmed by in situ hybridization on histological sections of TS diseased shrimps.

Animals↗
PubMed2001

Molecular epidemiology of respiratory syncytial virus.

OBJECTIVE: To determine the epidemiologic pattern of subgroups A and B and genotypes of respiratory syncytial virus (RSV) during two noncontinuous epidemics during 1990-1991 and 1997-1998 in Beijing. METHODS: Nasopharyngeal secretion (NPS) samples of RSV positive or RSV isolates tested by indirect immunofluorescence (IIF) assay were classified into subgroups A and B. Isolates of RSV were divided into at least six different lineages, designated NP1-NP6, by restriction mapping of the N gene. Np1, 3 and 6 were given by subgroup B isolates, while NP2, 4 and 5 were given by subgroup A isolates. Strains of subgroup A were further subdivided into six lineages SHL1-SHL6 on the basis of the SH gene sequence. SH lineages were closely related to each other and to NP1-NP6. Strains of SHL1, 3 and 4 were closely related and belonged to NP2, SHL2 and 6 to NP4, and SHL5 to NP5. RESULTS: Of 145 RSV NPS samples from the 1997-1998 epidemic, 83 (57.2%) were of subgroup B RSV positive, 62 (42.8%) of subgroup A RSV positive. The rate of occurrence of subgroup A to B strains was about 1:1.3. Two of 10 isolates during the epidemic were subgroup A strains, whereas 8 were subgroup B strains. The rate of occurrence of subgroup A to B strains was 1:4. Eight subgroup A strains of 10 isolates from the 1990-1991 epidermic were dominant; the proportion of subgroup A to B strains was 4:1. With 10 RSV isolates in 1997-1998, all 2 subgroup A strains gave N gene fragment restriction pattern NP4, and fell into SH lineage SHL2, whereas 8 subgroup B strains all belonged to NP3. All 8 subgroup A isolates from the 1990-1991 epidemic gave pattern NP4, and fell into SHL2, while 2 subgroup B strains all belonged to NP3. The classification of subgroups A and B deduced from NP patterns corresponded to the definition of these subgroups by monoclonal antibodies. CONCLUSIONS: These observations confirm that subgroups A and B or multiple lineages of RSV co-circulated in Beijing, but different genome types predominated each year. Moreover, very similar viruses were isolated up to more than 5 years ago, indicating that despite apparent diversity of the subgroup A strains, the separate lineages might be relatively stable.

Genotype↗
PubMed1999

Organization of the gene cluster for biosynthesis of penicillin in Penicillium nalgiovense and antibiotic production in cured dry sausages.

Several fungal isolates obtained from two cured meat products from Spain were identified as Penicillium nalgiovense by their morphological features and by DNA fingerprinting. All P. nalgiovense isolates showed antibiotic activity in agar diffusion assays, and their penicillin production in liquid complex medium ranged from 6 to 38 microgram. ml-1. We constructed a restriction map of the penicillin gene cluster of P. nalgiovense and found that the organization of the penicillin biosynthetic genes (pcbAB, pcbC, and penDE) is the same as in Penicillium chrysogenum and Aspergillus nidulans. The pcbAB gene is located in an orientation opposite that of the pcbC and penDE genes in all three species. Significant amounts of penicillin were found in situ in the casing and the outer layer of salami meat during early stages of the curing process, coinciding with fungal colonization, but no penicillin was detected in the cured salami. The antibiotic produced in situ was sensitive to penicillinase.

Blotting, Southern↗
PubMed1998

Evolution of mitochondrial DNA in yeast: gene order and structural organization of the mitochondrial genome of Saccharomyces uvarum.

We have determined the size, the restriction map and the gene order of the mitochondrial genome of the yeast Saccharomyces uvarum. Sequence analysis of the mitochondrial COXII gene confirmed the position of this yeast in the Saccharomyces cerevisiae-like group, near Saccharomyces cerevisiae and Saccharomyces douglasii. Most mitochondrial genes have been positioned on this approximately 57-kb long genome and three regions containing putative replication origins have been identified. The gene order of S. uvarum suggests that the mitochondrial genome of the S.cerevisiae-like yeasts could have evolved from an ancestral molecule, similar to that of S. uvarum, through specific genome rearrangements.

Cytosine↗