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J R Stringer

Publications and source records attributed to J R Stringer.

At least 73 records · Page 4Linked to original sources
PubMed1991

Repeated DNA in Pneumocystis carinii.

A 16-kb DNA fragment designated Rp3-1 and cloned from the genome of rat-derived Pneumocystis carinii was found to contain sequences that were repeated approximately 70 times per genome. The repeated sequences in Rp3-1 spanned at least 10.4 kb. Sequences in Rp3-1 were present on each of the 16 P. carinii chromosomes resolved by field inversion gel electrophoresis. Most of the P. carinii genomic sequences homologous to those in the Rp3-1 clone appeared to be as long as those in the Rp3-1 clone but were highly polymorphic with respect to restriction enzyme cleavage sites. The Rp3-1 DNA fragment appears to be a member of a family of large, degenerate, dispersed repeats.

Animals↗
PubMed1990

Pneumocystis carinii karyotypes.

Pulsed-field gel electrophoresis techniques were used to examine the chromosomes of Pneumocystis carinii isolated from laboratory rats and two human subjects. P. carinii organisms isolated from each of four rat colonies and from two patients each produced a distinct band pattern, but in all cases the bands ranged in size from 300 to 700 kilobase pairs. P. carinii from three rat colonies produced patterns containing 15 prominent bands. Of these 15 bands, 2 stained more intensely than would be expected of bands of their size, suggesting that the P. carinii haploid genome contains 17 to 19 chromosomes. Summing the molecular sizes of the bands and accounting for staining intensities suggested that the haploid genome of rat-derived P. carinii contains on the order of 10(7) base pairs. Human-derived P. carinii produced patterns containing 10 to 12 bands which appeared to be similar to the 15-band patterns seen in rat-derived P. carinii with respect to the size range of the bands. P. carinii from the fourth rat colony produced a more complex band pattern containing approximately 22 bands, most of which appeared to comigrate with the bands present in one of the 15-band P. carinii patterns, suggesting that these animals were simultaneously infected by two different varieties of P. carinii. Hybridization experiments using oligonucleotide probes specific for the P. carinii 18S rRNA gene supported this possibility. The band pattern of P. carinii derived from a given rat colony was generally stable over time. P. carinii band patterns were not strictly rat strain specific and appeared to be transferrable between animals housed in the same room.

Animals↗
PubMed1989

Pneumocystis carinii: sequence from ribosomal RNA implies a close relationship with fungi.

Pneumocystis carinii is the etiologic agent of a lethal pneumonia which occurs in patients with the acquired immune deficiency syndrome (AIDS) and other immunocompromised hosts. The basic biochemical and genetic characteristics of P. carinii are poorly understood and its taxonomic classification as a protozoan is uncertain. To address the taxonomic question, a method was developed for the extraction of total RNA from P. carinii. Denaturing agarose gel electrophoresis showed the two ribosomal RNA species of P. carinii to be similar in size to those of other lower eukaryotes, including Saccharomyces cerevisiae. Three portions of the small ribosomal RNA of P. carinii were sequenced by reverse transcription from oligonucleotide primers. Three hundred seventy-two nucleotides of sequence were obtained. The sequence derived from P. carinii RNA contained regions that previous phylogenetic studies have shown to be highly variable among species, as well as regions that are highly conserved. Comparison of the P. carinii sequence to corresponding sequences of organisms from other taxa showed the P. carinii sequence to be more similar to sequences from the fungi (Saccharomyces cerevisiae, Neurospora crassa, Candida albicans, and Cryptococcus diffuens) than to protozoan sequences. These data suggest that P. carinii is more closely related to fungi than to protozoa.

Acanthamoeba↗
PubMed1989

A tandem pair of Leishmania donovani cation transporting ATPase genes encode isoforms that are differentially expressed.

The second gene (ATPase 1b) of a tandem pair of cation transporting ATPases from Leishmania donovani was cloned and sequenced. The sequence of this gene was very similar to its upstream neighbor (ATPase 1a). Both genes contained a 2922 base open reading frame capable of encoding a protein of 974 amino acids. The genes differed at 34 nucleotide base positions, predicting 20 amino acid differences between the two peptides. These changes were clustered at the carboxy terminus with 15 changes occurring in the COOH-terminal 37 amino acids. However, these changes did not alter the highly charged nature of the carboxy terminus observed in ATPase 1a. The sequence was also conserved for 73 bases upstream of ATPase 1a and 1b but downstream conservation was limited to 15 bases beyond the termination codon. RNA from ATPase 1a was 5.2 kb and was present in both developmental forms of Leishmania. By contrast the ATPase 1b gene expressed a 5.75 kb transcript which was much more abundant in the amastigote form of Leishmania than in the promastigote form.

Adenosine Triphosphatases↗
PubMed1987

Structure and expression of a tandem gene pair in Leishmania donovani that encodes a protein structurally homologous to eucaryotic cation-transporting ATPases.

An oligonucleotide probe was used to clone a cation-transporting ATPase gene from the genome of Leishmania donovani. The nucleotide sequence of the gene contained a 2,922-base-pair open reading frame that was predicted to encode a 107,406-dalton protein composed of 974 amino acids. The predicted L. donovani protein contained all the structural and functional domains expected to be present in a cation-transporting ATPase of the aspartyl phosphate class. The nucleotide sequence encoding the ATPase gene was duplicated in tandem in the parasite genome. Partial sequenation of the second member of the tandem repeat, which lay 2 kilobase pairs downstream of the ATPase gene, indicated that it was either identical to the first gene or very closely related to it. RNA homologous to either the ATPase gene or its adjacent relative was 5 kilobases in size and was approximately equally abundant in both promastigote and amastigote forms of the organism.

Adenosine Triphosphatases↗
PubMed1986

RecA independent recombination of poly[d(GT)-d(CA)] in pBR322.

Short sequence tracts composed of alternating guanosine and thymidine nucleotide residues poly[d(GT)-d(CA)] carried in a derivative of pBR322 were recombinogenic in a recA host. Recombination brought about by poly[d(GT)-d(CA)] tracts displayed two interesting properties: (i) the reaction was quasi-sequence-specific in that while recombination usually occurred between two poly[d(GT)-d(CA)] tracts, recombination also occurred between sequences bordering the dinucleotide repeats. (ii) recombination was enhanced when two poly[d(GT)-d(CA)] tracts were clustered within 250 base pairs of each other, but not when the repeats were separated by 3 kilobase pairs. The mechanism by which poly[d(GT)-d(CA)] stimulated recombination remains to be determined, but the behavior of these sequences is consistent with the idea that general recombination in E. coli may involve formation of Z-DNA.

Base Composition↗
PubMed1985

Unequal homologous recombination between tandemly arranged sequences stably incorporated into cultured rat cells.

Cultured rat cells deficient in endogenous thymidine kinase activity (tk) were stably transformed with a recombination-indicator DNA substrate constructed in vitro by rearrangement of the herpes simplex virus tk gene sequences into a partially redundant permutation of the functional gene. The recombination-indicator DNA did not express tk, but was designed to allow formation of a functional tk gene via homologous recombination. A clonal cell line (519) was isolated that harbored several permuted herpes simplex virus tk genes. 519 cells spontaneously produced progeny that survived in medium containing hypoxanthine, aminopterin, and thymidine. Acquisition of resistance to hypoxanthine, aminopterin, and thymidine was accompanied by the rearrangement of the defective tk gene to functional configuration. The rearrangement apparently occurred by unequal exchange between one permuted tk gene and a replicated copy of itself. Recombination was between 500-base-pair tracts of DNA sequence homology that were separated by 3.4 kilobases. Exchanges occurred spontaneously at a frequency of approximately 5 X 10(-6) events per cell per generation. Recombination also mediated reversion to the tk- phenotype; however, the predominant mechanism by which cells escaped death in the presence of drugs rendered toxic by thymidine kinase was not recombination, but rather inactivation of the intact tk gene.

Animals↗
PubMed1985

Recombination between poly[d(GT).d(CA)] sequences in simian virus 40-infected cultured cells.

CVI cells were transfected with oversized simian virus 40 (SV40) genomes that could be reduced to packageable size by alternative homologous recombination pathways involving either two polydeoxyguanylic-thymidylic acid X polydeoxycytidylic-adenylic acid (poly[d(GT).d(CA)]; abbreviated hereafter as poly(GT)] tracts or two tracts of homologous SV40 sequence. Plaque-forming viruses rescued by this procedure were found to contain genomes formed by homologous and nonhomologous recombination events. Half of the viable viral DNA molecules recovered were the result of recombination between two tracts of poly(GT). Approximately 20% of the rescued viral genomes were produced by homologous recombination between tracts of SV40 DNA. Nonhomologous recombination involving SV40 sequences was also a major pathway of deletion, producing ca. 30% of the viral plaques. Tracts of poly(GT) generated by recombination were variable in length, suggesting that recombination between poly(GT) tracts was usually unequal. On a per-nucleotide basis, poly(GT) recombination occurred eight times more frequently than did recombination between homologous SV40 DNA. This eightfold difference is the maximum recombinatory enhancement attributable to poly(GT) sequences. Although DNA sequence analysis showed that tracts of poly(GT) generated by recombination retained the alternating G-T repeat motif throughout their length, the contribution of the nonhomologous pathway to poly(GT) recombination cannot be ruled out, and the relative proclivity of a given length of d(GT).d(CA) sequence to undergo homologous recombination is probably less than eight times greater than that of an SV40 sequence of the same length.

Animals↗
PubMed1982

Mutant of simian virus 40 large T-antigen that is defective for viral DNA synthesis, but competent for transformation of cultured rat cells.

A mutant was isolated which demonstrates that the transforming activity of simian virus 40 large T-antigen is separable from its function in viral DNA replication. The mutant, SVR9D, is nonconditionally defective for viral DNA synthesis, but competent at wild-type level for morphological transformation of cultured rat cells. The lytic growth defect in SVR9D is complemented by the simian virus 40 A gene product present in the transformed CV1 cell line, COS1. The lesion in SVR9D DNA was mapped genetically by marker rescue of plaque formation and localized to a 214-base-pair segment of the viral genome bounded by nucleotide numbers 4100 and 4314. DNA sequence analysis showed the mutation to be an adenine-to-guanine transition at nucleotide number 4178. This change predicts a lysine-to-glutamic acid amino acid change at residue number 214 of the mutant large T-antigen polypeptide.

Animals↗
PubMed1981

Integrated simian virus 40 DNA: nucleotide sequences at cell-virus recombinant junctions.

DNA fragments containing the integrated viral DNA present in the simian virus 40 (SV40)-transformed rat cell lines SVRE9 and SVRE17 were cloned in procaryotic vectors, and the DNA sequences linking SV40 and cell DNA were determined. Comparison of the DNA sequences at the SV40-cell junctions in SVRE9 and SVRE17 cells with those of a previously characterized viral insertion from SV14B cells shows that no specific viral or cellular sequences occur at SV40-cell junctions and that the cellular DNA sequences adjacent to integrated SV40 DNA do not display the direct repeat structure characteristic of transposons and retrovirus proviruses.

Animals↗
PubMed1979

Isolation and localization of herpes simplex virus type 1 mRNA.

Herpes simplex virus (HSV) DNA bound to cellulose has been used as a reagent to isolate viral mRNA for size analysis on denaturing agarose gels. Total viral polysomal polyadenylated RNA was isolated from cells late after infection when such RNA has sequences encoded by approximately 45% of the HSV DNA. This RNA has a size range of from 1.5 to greater than or equal to 8 kilobases, with certain sizes, such as 1.7 to 1.9 kilobases, being favored. We have used the restriction endonucleases HindIII and XbaI singly and together to generate various sized fragments covering the entire HSV-1 genome. These fragments have been bound to cellulose to allow isolation of HSV-1 mRNA annealing to different regions of the viral genome. Discrete sizes of viral mRNA are associated with certain regions of the genome, but the mRNA population hybridizing to even the smallest restriction fragments is complex. We used hybridization of size-fractionated RNA to Southern blots of restriction fragments of HSV-1 DNA generated by the BglII as well as HindIII and XbaI endonucleases to confirm the preparative hybridization data and to provide some overlap data for positioning transcripts. The data of blot and preparative hybridization agreed very well and were combined to construct a preliminary transcription map of HSV-1. Such a map revealed at least two areas of the long unique region of the HSV-1 genome which annealed to a large number of HSV-1 transcripts. Furthermore, discrete-sized mRNA species larger than 5 kilobases in length were found only in the middle of the long unique region. The implications of these data are discussed.

DNA Restriction Enzymes↗
PubMed1979

Isolation and localization of herpes simplex virus type 1 mRNA abundant before viral DNA synthesis.

Herpes simplex virus type 1 (HSV-1) DNA covalently bound to cellulose was used as a reagent to isolate viral RNA transcripts for size analysis on denaturing agarose gels. Nuclear and polyribosomal RNA isolated at 2 h postinfection (p.i.) migrated with sizes between 1,500 and 5,500 nucleotides. At 6 h p.i. (when viral DNA synthesis is underway), viral polyribosome-associated polyadenylated RNA showed different discrete sizes of species predominating, with RNA larger than 5,500 nucleotides clearly present. Nearly 50% of the newly made viral RNA found in the nucleus at 6 h p.i. was from 5,000 to 10,000 nucleotides in length. A high-resolution transcription map of the viral mRNA abundant at 2 h p.i. was compiled from the hybridization of Southern blots of HSV-1 DNA restriction fragments to both sizes of fractionated polyribosomal polyadenylated RNA and 3' complementary DNA probe made to this size of fractionated RNA. We have identified and mapped 16 mRNA species abundant at 2 h p.i. These RNAs range in size from 1,500 to 5,300 nucleotides and map throughout the HSV-1 genome. In some instances, a direction of transcription can be suggested. Further, about one-third of this number of mRNA's has been found in cells infected with a DNA-negative temperature-sensitive mutant (tsB2) and grown at the nonpermissive temperature (39 degrees C).

Cell Nucleus↗
PubMed1978

Mapping early transcripts of herpes simplex virus type 1 by electron microscopy.

RNA displacement loop patterns in intact herpes simplex virus DNA and herpes simplex virus DNA restriction fragments indicate that viral RNA associated with polyribosomes early after infection is transcribed from three major areas of the genome. One area of early transcription is in the short segment of the viral DNA and is roughly delineated by the inverted repeat sequences bounding this segment. The other two areas of early mRNA transcription map in the long segment. Each of three major areas of early mRNA transcription can be further resolved into several regions of freqent looping bordered by regions in which RNA displacement loops are rare. These regions range in size from about 1.5 kilobases to about 9 kilobases with a mean size of about 3.5 kilobases. Although the data do not allow precise assignment of individual early gene locations, it is seen, even at the lowest level of resolution, that the early genes are not completely contiguous but are distributed along the length of the herpes simplex type 1 viral genome.

DNA Restriction Enzymes↗
PubMed1977

Quantitation of herpes simplex virus type 1 RNA in infected HeLa cells.

We have quantitatively analyzed the size and amount of herpes simplex virus (HSV)-specific RNA synthesized in HeLa cells using DNA and RNA excess hybridization. At 2 h after infection (early), transcripts from 20% of the total HSV DNA are present on polyribosomes as poly(A+) RNA. At this time, viral poly(A+) RNA comprises 60 to 75% of the newly synthesized poly(a+) mRNA on polyribosomes. By 6 h after infection (late), poly(A+) HSV RNA transcribed from 35 to 40% of the viral DNA is found on polyribosomes. These viral poly(A+) transcripts comprised as much as 90% of newly synthesized poly(A+) mRNA and are measurably larger than viral poly(A+) transcripts isolated early. Some but not all of this size difference is due to the fact that the poly(A) tails on early transcripts are shorter than those found on transcripts made late. Even late after infection, a small but readily measurable amount of cellular poly(A+) RNA is still being made and entering polyribosome complexes. In the nucleus, late after infection, poly(A+) HSV RNA is complementary to 50% of the total HSV DNA. Both early and late after infection, total nuclear viral transcripts are, on the average, larger than viral transcripts found on polyribosomes; however, nuclear HSV poly(A+) RNA is not measureably larger than the corresponding cytoplasmic viral poly(A+) sequences at either time. A major portion (30 to 40%) of the polyribosomal HSV RNA made either early or late after infection is not polyadenylated. This HSV poly (A-) RNA is transcribed from the same sequences as HSV poly(A+) RNA but, when labeled and isolated either early or late after infection, both nuclear and polyribosomal viral poly(A-) RNA molecules sediment faster in sucrose-formaldehyde gradients than their polyadenylated counterparts.

Base Sequence↗
PubMed1977

5'-Terminal and internal methylated nucleosides in herpes simplex virus type 1 mRNA.

RNA labeled with [methyl-3H]methionine and/or [32P]orthophosphate was isolated from the polyribosomes of herpes simplex virus (HSV) types 1-infected cells and separated into polyadenylylated [poly(A+)]and non-polyadenylylated [poly(A-)] fractions. Virus-specific RNA was obtained by hybridization in liquid to either excess HSV DNA or filters containing immobilized HSV DNA. Analysis in denaturing sucrose gradients indicated that HSV-specific poly(A+) RNA sedimented in a broad peak, with a modal S value of 20. The ratio of [3H]methyl to 32P decreased with increasing size of RNA, suggesting that each RNA chain contains a similar sumber of methyl groups. Further analysis indicated an average of one RNase-resistant structure of the type m7G(5')pppNmpNp or m7G(5')pppNmpNmpNp per 2,780 nucleotides. The following components were identified in the 5'-terminal oligonucleotides of polyribosome-associated HSV-specific poly(A+) and poly(A-) RNA: 7-methylguanosine, N6,2'-O-dimethyladenosine, and the 2'-O-methyl derivatives of guanosine, adenosine, uridine, and denosine, and the 2'-O-methyl derivatives of guanosine, adenosine, uridine, and cytidine. The most common 5'-terminal sequences were m7G(5')pppm6Am and m7G(5')pppGm. An additional modified nucleoside, N6-methyladenosine, was present in an internal position of HSV-specific RNA.

Adenosine↗