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PubMed1983

Extrachromosomal deer fibromavirus DNA in deer fibromas and virus-transformed mouse cells.

The non-virus-producing fibromatous portions of five deer fibromas were examined for deer fibromavirus (DFV) DNA sequences. Liquid-phase hybridization revealed 100 to 330 copies per cell of the virus genome. Southern blot analysis of undigested deer tumor DNA preparations indicated that most of the DFV DNA was present as monomeric, unintegrated genomes; however, restriction enzyme digestion patterns suggest a small population of resistant DFV sequences. DFV DNA was also present in virus-transformed NIH/3T3 mouse cells as multiple, extrachromosomal genomes.

Animals↗
PubMed1992

Extrachromosomal human immunodeficiency virus type 1 sequences are methylated in latently infected U937 cells.

Long-term human immunodeficiency virus type 1 (HIV-1) infection of the human monocytic cell line U937 resulted in a progressive loss of infectivity that was correlated with the accumulation of stable, extrachromosomal forms of viral DNA. Viral latency was also characterized by reduced levels of HIV-1 transcription. The structure and activity of extrachromosomal viral DNA (E-DNA) in a fully latent U937 cell line was investigated by molecular cloning and DNA transfection. The resulting 18-kb E-DNA clone was composed of an intact HIV-1 sequence flanked by 7 kb of host sequence to one side and 1 kb of host DNA to the other side. This configuration is the result of retroviral integration into a highly repetitive element of the Alu family. Transfection of the E-DNA clone resulted in the production of infectious virus, indicating that viral latency was not the result of mutations in the HIV-1 genome. Analysis of CCGG sites revealed extensive de novo methylation of viral sequences present within E-DNA. These results suggest that modification of extrachromosomal viral DNA sequences is a mechanism for HIV-1 latency in long-term infected U937 cells.

Base Sequence↗
PubMed1975

Studies of mouse mitochondrial DNA in Escherichia coli: structure and function of the eucaryotic-procaryotic chimeric plasmids.

The mouse mitochondrial DNA genome has been cloned in Escherichia coli by linking it to the pSC101 plasmid replicon at cohesive-ended cleavage sites generated by Eco Rl restriction endonuclease. The four possible configurations of chimeric molecules that contain the nucleotide sequences of mitochondrial DNA in their native relationship were distinguished by Hind III restriction endonuclease digestion and electron microscopic heteroduplex analysis. Chimeric molecules utilize the pSC101 replication origin and do not maintain the "D-loop" region or the low frequency of ribonucleotides found in native mitochondrial DNA. Hybridization of the RNA synthesized in E. coli minicells carrying the four types of chimeras indicates that transcription occurs predominately on the light strand of the mitochondrial DNA in all cases. This result implies that initiation of RNA synthesis occurs within the mitochondrial DNA segment. Although specific polypeptide synthetis is directed by the mitochondrial DNA segment of each of the chimeras in E. coli minicells, the molecular weight distribution of the polypeptides synthesized consists primarily of low molecular weight species and thus differs from that observed in mitochondria in mouse L cells.

Chimera↗
PubMed1975

Characterization of six cloned DNAs from Drosophila melanogaster, including one that contains the genes for rRNA.

pDm plasmids were constructed from D. melanogaster and pSC101 DNAs by a modification of the EcoR1-ligase method which insured that each hybrid molecule contained a single segment of D. melanogaster chromosomal DNA (Dm segment). The sequences in the Dm segments of six cloned pDm DNAs were mapped within the D. melanogaster polytene chromosomes by in situ hybridization, and their repetition frequencies within the Dm segment and within the genome were determined. Four of these segments consist of sequences that are confined to single chromomeric regions in the polytene chromosomes and exhibit little or no repetition. The characteristics of this group, and also two of three Dm segments analyzed earlier (Wensink et al., 1974), are inconsistent with tandem repetition models of the chromomere. By contrast, the other two Dm segments contain moderately repetitive sequences that are located in the heterochromatin. One of these appears to be a segment of the Y chromosome in which about half the sequences are nonrepetitive and half are repeated about 33 times per genome, though they are not repeated within the segment. The second contains the DNA coding for 18 and 28S rRNA.

Animals↗
PubMed1976

Amplification and characterization of a beta-globin gene synthesized in vitro.

Full-length, double-stranded globin DNA was synthesized in vitro starting from rabbit globin mRNA. Several restriction endonuclease cleavage sites with known recognition sequences were mapped on this DNA as a means of assessing the accuracy of in vitro synthesis. By comparing this map with the nucleotide sequences known or predicted from the amino acid sequences of alpha-and beta-chain rabbit hemoglobin, it was possible to show that the synthetic globin DNA is a faithful copy of beta-globin mRNA. Amplification of the synthetic globin DNA was achieved by inserting the molecule into the plasmid PMB9 using the poly(dA)-(dT) joining procedure, and transforming E. coli with the hybrid DNA. Transformants carrying beta-globin DNA were identified by colony hybridization using purified 125I-beta-mRNA probe. Comparison of the restriction maps of the synthetic and inserted globin DNAs showed that the entire synthetic globin DNA molecule was amplified without sequence rearrangements. Both the synthetic and the cloned DNA include the entire coding sequence of the beta-globin gene plus a substantial portion of the untranslated regions flanking the structural gene.

Base Sequence↗
PubMed1976

A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome.

Using the poly(dA-dT) "connector" method (Lobbanand Kaiser, 1973), a population of annealed hybrid circular DNAs was constructed in vitro; each hybrid DNA circle contained one molecule of poly(dT)-tailed Col El-DNA (LRI) annealed to any one of a collection of poly(dA)-tailed linear DNA fragments, produced originally by shearing total E. coli DNA to an average size of 8.5 x 10(6) daltons. This annealed DNA preparation (12 mug) was used to transform an F+ recA E. coli strain (JA200), selecting transformants by their resistance to colicin El. A collection or "bank" pf pver 2000 colicin El-resistant clones was thereby obtained, 70% of which were shown to contain hybrid Col El DNA (E. coli) plasmids. This colony bank is large enough to include hybrid plasmids representative of the entire E. coli genome. Individual plasmids have been readily identified by replica mating the collection onto plates seeded with cultures of various F- auxotrophic recipients, selecting for complementation of the auxotrophic markers by F-mediated transfer of hybrid plasmids to the F- recipients. In this manner, over 80 hybrid Col El-DNA (E. coli), plasmid-bearing clones have been identified in the colony bank, and about 40 known E. coli genes have been tentatively assigned to these various plasmids. The hybrid plasmids are transferred efficiently from F+ donors to appropriate F- recipients. The use of this method to establish similar colony banks in E. coli containing hybrid plasmids representative of various simple eucaryotic genomes is discussed.

Chromosome Mapping↗
PubMed1976

Histone genes of the sea urchin (S. purpuratus) cloned in E coli: order, polarity, and strandedness of the five histone-coding and spacer regions.

Sea urchin (S. purpuratus) histone DNA of constructed plasmid chimeras cloned in E. coli was cleaved with the restriction endonucleases Eco RI, Hind III, Sal I. Bam I, and Hha I. The resulting fragments were ordered and isolated directly from agarose gels or cloned into other plasmids. Each fragment hybridized to one or another of the five histone mRNAs and elucidated the order of the histone genes in each of the cloned fragments. Some DNA did not hybridize to histone mRNAs and was identified as spacer DNA located between coding regions. Total sea urchin DNA was cleaved with restriction endonucleases, fractionated on agarose gels, and hybridized to histone mRNAs or histone DNA. The results revealed the order of the five histone genes in the histone gene repeat unit and demonstrate that the histone spacer DNA have little sequence homology to other genes. ExonucleaseIII digestion of specific linear chimeric histone DNA plasmids followed by hybridization with mRNAs demonstrated the existence of all five histone genes on one strand of DNA and the 5'-3' polarity of that strand. These results, in conjunction with the data of Wu et al. (1976), allow us to construct a map of coding and spacer sequences in the transcribed strand of S. purpuratus histone gene repeat unit: (see article).

Animals↗
PubMed1976

The relative positions of sea urchin histone genes on the chimeric plasmids pSp2 and pSp17 as studied by electronmicroscopy.

The relative positions of the sea urchin histone genes and the spacer regions on the chimeric plasmids pS p2 and pSp17 have been mapped by hybridizing total histonemessenger RNA to single strands of the plasmid DNAs. The lengths and spacing between the several RNA:DNA duplex regions on the single strands of DNA were measured by the gene 32-ethidium bromide electron microscope mapping method. We find that the genes are interdigitated with spacer sequences of different lengths; that there are three coding sequences on pSp2, all on the same strand, with the relative order H1, H4, and B4; and that there are two coding sequences on pSp17, both on the same strand, corresponding to the messages denoted B1 and B2-B3, where B4, B1, and B2-3 are electrophoretically resolved components of histone mRNA, all of size intermediate between the larger H1 and the smaller H4 message.

Chimera↗
PubMed1976

Recombination between bacterial plasmids leading to the formation of plasmid multimers.

We report here the formation of plasmid multimers in E. coli. Multimers are covalently closed, circular molecules composed of tandem repeats of the monomer plasmid. Multimer formation occurs at high frequency in rec+, recB-C-, and recF- hosts. Multimer formation is not detected in recA- hosts and occurs at reduced frequency in recB-C-F- hosts. We conclude that multimer formation is mediated by a single reciprocal recombination event occurring at homologous regions on the two plasmids.

Coliphages↗