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Biomedical subjects

A Honigman

Publications and source records attributed to A Honigman.

At least 37 records · Page 2Linked to original sources
PubMed1989

Translation of gag, pro, and pol gene products of human T-cell leukemia virus type 2.

Sequence analysis of human T-cell leukemia proviral DNA revealed three open reading frames arranged at a -1 position relative to one another. On the basis of homology to other retroviruses, these open reading frames were assigned to the gag, pro, and pol genes. To characterize the primary protein products of these genes and their modes of synthesis, a DNA clone of human T-cell leukemia virus type 2 was transcribed and translated in vitro. Analysis of the viral proteins revealed three polyproteins with molecular masses of 58, 75, and 112 kilodaltons at relative frequencies of 100:13:0.9, respectively. These proteins were mapped on the viral genome by both internal deletions and 3'-end truncations at gag, pro, and pol, respectively. The results indicate that translation of the pol gene requires two independent frameshift events, and the readthrough frequencies at the two frameshift sites appeared to be similar.

Chromosome Mapping↗
PubMed1986

Transcription termination and processing sites in the bacteriophage lambda pL operon.

S1 nuclease mapping was performed on transcripts from the major leftward operon of the bacteriophage lambda in order to locate the 3' ends of stable RNA species produced in vivo. The analysis was carried out on RNA purified from either an induced lambda prophage or bacteria carrying a plasmid containing a large segment of lambda including the intact PL operon through the bet gene. The S1 nuclease mapping was performed on transcripts produced in the presence and the absence of the N antitermination function, and in the presence and the absence of either the RNase III processing enzyme or the Rho factor. The results of this work indicate that the intercistronic region between the N and ral genes of lambda contains three sites at which transcripts end under N-Rho+ conditions (positions on the lambda sequence: 34,826, 34,558 and 34,393). The distal two correspond to the two sites previously described in this region as tL1 (on both sides of the BamHI site). In the region between ral and Ea10, we mapped the 3' ends of three species of RNA. The 3' end of one species was found to be located 90 nucleotides proximal to tL2a, at 34,000 in the lambda sequence. The terminator at this site may be partially N-resistant. In an RNase III deficient host, an additional RNA species is formed. The 3' end of this RNA species is located at tL2a (33,910 on the lambda sequence). In the presence of the antitermination N gene product, the readthrough transcripts are processed to form a 3' end at position 33,980 on the lambda sequence. These results suggest that elongation of transcription of the lambda PL operon is reduced gradually by clusters of termination located between genes and that the expression of the terminated products is further controlled by processing of the mRNA.

Bacteriophage lambda↗
PubMed1986

Cloning and expression of the lepidopteran toxin produced by Bacillus thuringiensis var. thuringiensis in Escherichia coli.

The Bacillus thuringiensis var. thuringiensis strain 3A produces a proteinaceous parasporal crystal toxic to larvae of a variety of lepidopteran pests including Spodoptera littoralis (Egyptian cotton leaf worm), Heliothis zeae, H. virescens and Boarmia selenaria. By cloning of individual plasmids of B. thuringiensis in Escherichia coli, we localized a gene coding for the delta-endotoxin on the B. thuringiensis plasmid of about 17 kb designated pTN4. Following partial digestion of the B. thuringiensis plasmid pTN4 and cloning into the E. coli pACYC184 plasmid three clones were isolated in which toxin production was detected. One of these hybrid plasmids pTNG43 carried a 1.7-kb insert that hybridized to the 14-kb BamHI DNA fragments of B. thuringiensis var. thuringiensis strains 3A and berliner 1715. This BamHI DNA fragment of strain berliner 1715 has been shown to contain the gene that codes for the toxic protein of the crystal (Klier et al., 1982). No homologous sequences have been found between pTNG33 and the DNA of B. thuringiensis var. entomocidus strain 24, which exhibited insecticidal activity against S. littoralis similar to that of strain 3A.

Antigens, Bacterial↗
PubMed1985

Plasmid vectors designed for the analysis of transcription termination signals.

We have constructed synthetic operons in which two genes (cat and lacZ or cat and galK) were placed in tandem under the control of the bacteriophage lambda oLpL operator and promoter. Restriction sites were introduced between the promoter and the proximal cat gene or between the cat and lacZ or galK genes. In the latter case, introduction of a transcriptional terminator between the two structural genes should affect only the distal gene. Thus, following induction, the expression of the cat gene serves as an internal control, compensating for changes due to plasmid copy number or possible decrease in transcription initiation. We used these plasmids to select a lambda DNA fragment which includes the N-unresponsive tJ transcriptional terminator. This DNA fragment was inserted between the cat and galK genes. Enzymatic assays of these two gene activities following induction indicate that transcripts initiated at the pL promoter under N+ conditions terminate at tJ between the two genes. S1-nuclease analysis showed that these transcripts terminate at several sites in the tJ region. Similar results were obtained whether the host cells were RNaseIII+ or RNaseIII-. As a control, we showed a complete antitermination of the lambda t'I terminator under similar conditions, indicating that a sufficient amount of the N gene product is made from one N gene copy to suppress terminators carried on multicopy plasmids.

Acetyltransferases↗
PubMed1985

Generation of a uniform 3' end RNA of murine leukemia virus.

Using the S1 nuclease mapping technique, we demonstrated that the majority of Moloney murine leukemia RNA molecules, isolated either from the nucleus or cytoplasm of infected mouse cells, share a uniform 3' end located at the border of the R and U-5 regions of the long terminal repeat. When the long terminal repeat sequences were inserted in the pSV plasmid downstream of the simian virus 40 late promoter, the 3' end of the viral RNA was also generated close to the R region of the long terminal repeat. These results demonstrate that the long terminal repeat signals the generation of an authentic 3' end when situated downstream of an actively transcribed region.

Animals↗
PubMed1984

Cloning of L-2 DNA in Escherichia coli pOL4 plasmid.

A physical map of L-2 DNA was constructed using restriction endonucleases. Based on this map the five HincII-generated L-2 DNA fragments (A-E) were cloned into the SmaI site of Escherichia coli vector plasmid pOL4, that was designed to analyze promoters and transcriptional terminators. The insertion of the HincII-generated L-2 DNA fragments into this plasmid clearly demonstrated that a fragment (fragment E) with a size of 1.1 kbp carried a sequence that initiated transcription in E. coli.

Acholeplasma↗
PubMed1983

A unique sequence in murine leukemia virus long terminal repeat functions as a termination signal for transcription in Escherichia coli.

Moloney murine leukemia virus DNA fragments were subcloned into two plasmids downstream of active promoters to detect termination sequences for transcription initiated at these promoters. Two of the viral DNA fragments (2,050 and 595 base pairs) were able to block transcription when inserted in one orientation but not when inserted in the other. These two viral DNA fragments contained long terminal repeat sequences. Deletion of 84 base pairs from the U-3 and R regions in the long terminal repeat removed the transcription termination activity.

Cloning, Molecular↗
PubMed1982

The use of the plasmid pHA10 in the isolation of lambda PL promoter mutations.

The plasmid pHA10 provides a good positive selection system for PL promoter mutations. It contains the lambda EcoR1-D fragment cloned into the pBR322 plasmid. Inactivation of the temperature sensitive lambda repressor at 42 degrees C leads to transcription of the kil gene and host death. One group of survivors at 42 degrees C is saved due to mutations in the PL promoter. After several screening procedures one such mutant was isolated. Restriction enzyme analysis of the plasmid DNA followed by sequence determination showed it to be a 24 base pair deletion beginning half-way through the "Pribnow box" continuing through part of the -35 region. It removes the OL1 section of the OL operator plus one base pair of OL2. This mutation (called dpl) may be useful to the study of the lambda OLPL regulation system. The effect of base changes within the PL promoter on the promoter activity are discussed.

Bacteriophage lambda↗
PubMed1981

Plasmid vectors for positive selection of DNA inserts controlled by the lambda pL promoter, repressor and antitermination function.

Hybrid plasmids consisting of pBR322 or pOP203-3 and the EcoRI-D fragment of lambda DNA kill their bacterial host upon expression of a lambda gene (probably the kil function) located either between or across the SalI sites. The plasmids from surviving hosts are acquired deletions that remove the lambda kil gene or insertions that block the transcription of the kil gene. Some plasmids probably carry point mutations. Based on these findings, we constructed two vector plasmids, pKL1 and pHA10, which can be used for a direct positive selection of cloned fragments. These plasmids are particularly useful for the cloning and selection of N-unresponsive termination signals using BamHI and its isoschizomers. The DNA fragments cloned into these plasmids are under control of the strong pL promoter, which can be regulated by the lambda repressor, and the antitermination activity of the N gene product.

Bacteriophage lambda↗
PubMed1981

Cloning and characterization of a transcription termination signal in bacteriophage lambda unresponsive to the N gene product.

The pHA10 plasmid was designed for the cloning and selection of transcriptional termination signals. This study demonstrates the use of pHA10 as a cloning vehicle in the selection and characterization of an N-unresponsive terminator. Following the random cloning of lambda DNA, and N-unresponsive transcription terminator was isolated. Hybridization of in vivo 32P-labeled RNA to various DNA fragments, using the Southern (1975) blotting technique, revealed that the transcriptional barrier that cannot be overridden in the presence of N is located close to the end of gene J of lambda DNA. The terminator determines the 3'-end of the pL operon and prevents the transcription of the anti-sense DNA strand of lambda late genes. The primary structure and other characteristics of this terminator are now under investigation.

Bacteriophage lambda↗
PubMed1980

Identification of palindromic sequences recognized by restriction endonucleases, as based on the tabularized sequencing data for seven viral and plasmid DNAs.

Computer search of DNA sequences for phages phi X174, G4, M13 and fd, plasmids pBR322 and pAO3, and virus SV40, was employed to prepare tables specifying the size classes and frequencies of DNA segments located between all possible tetra-, penta- and hexanucleotide palindromes. As described earlier (Fuchs et al., 1978), these tables permit identifying sequences recognized by most of the restriction endonucleases. The effect of sequencing errors on the accuracy of the present identification method is evaluated. Only four of the 224 listed sequences do not appear in any of the seven DNAs, leading to discussion (see Appendix) on the natural sequence distribution.

Bacteriophage phi X 174↗
PubMed1978

A simple method for identifying the palindromic sequences recognized by restriction endonucleases: the nucleotide sequence of the AvaII site.

Tables specifying the frequencies, distances between and positions of all possible tetra-, penta- and hexanucleotide palindromes in phiX174 and SV40 viral DNAs were prepared by a computer search of their base sequences. A simple method based on these tables is described for identifying the sequence recognized by any specific restriction endonuclease. The method requires experimental determination of the number and approximate sizes of the fragments obtained by digestion of phiX174 RF and SV40 DNAs. Using this method we identified the sequence for AvaII restriction endonuclease as 5'-GG(AT)CC.

Base Sequence↗
PubMed1976

4S oop RNA is a leader sequence for the immunity-establishment transcription in coliphage lambda.

oop RNA, which is initiated at the po promoter and is 81 nucleotides long, can function as a leader sequence for the lambda immunity establishment transcription, previously believed to originate at a special promoter pre located in the y region. Thus, oop RNA seems to have a dual role, either favouring the lytic cycle as a primer for the initiation of lambda DNA replication, or leading to the establishment of lysogeny when elongated into the imm transcript, which directs synthesis of the repressor.

Coliphages↗
PubMed1975

A pleiotropic regulatory mutation in lambda bacteriophage.

Lambda bacteriophage mutants, lambdasar, were isolated. These mutants can form plaques on a non lysogenic lawn and are unable to grow on nonimmune (imm-), cro constitutive hosts. Analysis of the restriction of lambdasar by a set of defective lysogens suggested that both the cro and cII gene products participate in the inhibition. The sar mutations were mapped in the ori region between the genes cII and O. Complementation experiments showed that under the restrictive conditions lamdasar is defective in the expression of both the N and the O genes. Transcription analyses support these findings, as lambdasar is unable to serve as a template for transcription after infecting cro constitutive hosts. In addition lambdasar does not replicate under the restrictive conditions, although its DNA can bind to the host membrane to some extent. The Sar phenotype can be relieved by removing sites of action of cro either by a V2 mutation or by substituting the lambda immunity region by imm434 or imm21. Similarly introducing a cy mutation, which interferes with the action of the cII gene product, also eliminates the Sar effect. The sar mutation can suppress cy mutations as manifested in plaque morphology, lysogenization frequency, cI repressor synthesis and the expression of rex function. Suppression takes place only when the sar mutation is present in cis to cy and it requires the action of the cII and cIII gene products. It is suggested that the sar mutation suppresses cy by activating a new promoter for repressor synthesis, pro. The results also suggest that the cII and cIII gene products may act at a site other than y.

Chromosome Mapping↗