Showing papers on "Restriction map published in 1980"

A restriction map of the bacteriophage T4 genome.

Abstract: We report a detailed restriction map of the bacteriophage T4 genome and the alignment of this map with the genetic map The sites cut by the enzymes BglII, XhoI, KpnI, SalI, PstI, EcoRI and HindIII have been localized Several novel approaches including two-dimensional (double restriction) electrophoretic separations were used

Organization of the recA gene of Escherichia coli

Abstract: The restriction map of a BamHI DNA fragment that contains the recA gene of Escherichia coli has been established and a large portion of the fragment's nucleotide sequence has been determined. The coding region of the recA gene contains 1059 nucleotide residues and encodes a single protein of 353 amino acid residues. The amino acid sequence of the first five residues of the NH2 terminus of the recA protein agrees with the sequence predicted from the DNA sequence except for the absence of formylmethionine in the purified protein. Immediately after the coding sequence, there is a G+C-rich sequence with dyad symmetry followed by an A+T-rich sequence. These could signal termination of transcription. The site of initiation for synthesis in vitro of the recA messenger RNA has been determined by analysis of the 5' nucleotide sequence of [gamma-32P]ATP-labeled transcripts. The promoter region shos a high degree of symmetry and contains sequences commonly found in recognition and binding sites for RNA polymerase.

Isolation and characterization of the mouse metallothionein-I gene.

Abstract: Double-stranded cDNA was synthesized from a mouse liver mRNA fraction enriched for metallothionein mRNA activity, ligated to restriction site linkers, inserted into pBR322, and used to transform Escherichia coli chi 1776. The sequence of the largest plasmid containing DNA that hybridized to metallothionein mRNA was determined and shown to contain a 380-base-pair insert that includes the entire coding region and 3' untranslated region of metallothionein-I. The metallothionein-I insert was nick-translated and used to screen both a mouse myeloma and a mouse embryo DNA library in bacteriophage lambda. A metallothionein-I genomic clone containing 13-15 kilobase pairs of mouse DNA was isolated from each library. Both contain a 3.8-kilobase-pair EcoRI fragment that hybridizes to the metallothionein-I probe. The location, size, and orientation of the metallothionein-I gene within the 3.8-kilobase-pair fragment were determined by heteroduplex and restriction mapping. The gene spans 1.1 kilobase pairs and contains at least two introns.

New map of bacteriophage lambda DNA.

Abstract: A map of bacteriophage lambda was constructed, including accurate positions for all 41 cut sites made by 12 different restriction enzymes Over 100 fragments from single, multiple, and partial enzyme digestions were measured versus standards that were calibrated with respect to DNA molecules of known sequence The data were subjected to least-squares analysis to assign map coordinates In no case did a fragment size predicted from the map differ from the measurement of the fragment by more than +/- 5% This low error rate was consistent in all size ranges of fragments The total length of lambda was calculated as 49,133 nucleotide pairs This probably is accurate to within 500 base pairs

Molecular cloning of the avian erythroblastosis virus genome and recovery of oncogenic virus by transfection of chicken cells.

Abstract: Avian erythroblastosis virus (AEV) causes erythroblastosis and sarcomas in birds and transforms both erythroblasts and fibroblasts to neoplastic phenotypes in culture. The viral genetic locus required for oncogenesis by AEV is at present poorly defined; moreover, we know very little of the mechanism of tumorigenesis by the virus. To facilitate further analysis of these problems, we used molecular cloning to isolate the genome of AEV as recombinant DNA in a procaryotic vector. The identity of the isolated DNA was verified by mapping with restriction endonucleases and by tests for biological activity. The circular form of unintegrated AEV DNA was purified from synchronously infected quail cells and cloned into the EcoRI site of lambda gtWES x B. A restriction endonuclease cleavage map was established. By hybridization with complementary DNA probes representing specific parts of avian retrovirus genomes, the restriction map of the cloned AEV DNAs was correlated with a genetic map. These data show that nucleotide sequences unique to AEV comprise at least 50% of the genome and are located approximately in the middle of the AEV genome. Our data confirm and extend previous descriptions of the AEV genome obtained by other procedures. We studied in detail two recombinant clones containing AEV DNA: the topography of the viral DNA in the two clones was virtually identical, except that one clone apparently contained two copies of the terminal redundancy that occurs in linear viral DNA isolated from infected cells; the other clone probably contained only one copy of the redundant sequence. To recover infectious virus from the cloned DNA, we developed a procedure for transfection that compensated for the defectiveness of AEV in replication. We accomplished this by ligating cloned AEV DNA to the cloned DNA of a retrovirus (Rous-associated virus type 1) whose genome could complement the deficiencies of AEV. Ligation of the two viral DNAs was facilitated by using a neutral fragment of DNA as linker between otherwise noncompatible termini. Cloned AEV DNA gave rise to infectious AEV capable of transforming fibroblasts and bone marrow cells in culture and of inducing both sarcomas and erythroleukemia in chickens. We conclude that the cloned DNAs represent the authentic genome of AEV undisturbed by the cloning procedure. Molecular cloning offers a powerful approach to the identification and characterization of retrovirus genomes.

Isolation of DNA from agarose gels using DEAE-paper. Application to restriction site mapping of adenovirus type 16 DNA

Abstract: A new method for isolating DNA from agarose gels is described. The method involves the simultaneous transfer of all DNA-fragments from an agarose slab gel onto DEAE-cellulose paper and the elution of the individual fragments from the paper with 1 M NaCl. DNA isolated from agarose gels in this way is susceptible to cleavage with several restriction endonucleases, and can be labeled in vitro with E coli DNA-polymerase I, T4 DNA-polymerase and T4 polynucleotide kinase. We have used the method to construct restriction endonuclease maps of adenovirus type 16 DNA.

Localization of the plasmid (pKM101) gene(s) involved in recA+lexA+-dependent mutagenesis.

Abstract: Twenty Tn5 insertion mutants of the drug resistance plasmid pKM101 have been isolated that are unable to enhance mutagenesis with ultraviolet (UV) irradiation or methyl methanesulfonate. By restriction mapping, the Tn5 insertion in each of these pKM101 mutants was shown to be within a 1.9 kb region of the plasmid genome. We have termed this segment of the pKM101 map the muc (mutagenesis: UV and chemical) gene(s). Characterization of these mutants indicated that any Tn5 insertion within the muc gene(s) abolished the ability of pKM101 to: (a) enhance spontaneous, UV and chemical mutagenesis, (b) increase host survival following UV-irradiation, (c) increase the survival of UV-irradiated phage plated on irradiated or unirradiated cells, and (d) suppress the repair and mutagenesis deficiencies of a umuC − mutant. Possible models to explain the role of the pKM101 muc gene(s) in mutagenesis and repair are discussed.

Genetic organization of the broad-host-range IncP-1 plasmid R751.

Abstract: We have identified regions encoding conjugal transfer, plasmid maintenance, and trimethoprim resistance on the IncP-1 plasmid R751 by complementation tests with cloned deoxyribonucleic acid fragments and self-replicating derivatives constructed in vitro. The genes for replication and transfer show a scattered organization similar to that previously determined for RK2, another IncP-1 plasmid. Derivatives of RK2 are able to complement R751 derivatives defective in these functions. Restriction enzyme cleavage sites in R751 deoxyribonucleic acid are clustered in regions of the plasmid physical map. Neither region is required for plasmid maintenance or transfer, although one determines resistance to trimethoprim. A similar clustering of cleavage sites is seen with RK2, which nevertheless has a very different restriction map.

Defective and plaque-forming lambda transducing bacteriophage carrying penicillin-binding protein-cell shape genes: genetic and physical mapping and identification of gene products from the lip-dacA-rodA-pbpA-leuS region of the Escherichia coli chromosome.

Abstract: A series of defective lambda transducing phage carrying genes from the lip-leuS region of the Escherichia coli chromosome (min 14 on the current linkage map) has been isolated. The phage defined the gene order as lac---lip-dacA-rodA-pbpA-leuS---gal. These included the structural genes for penicillin-binding protein 2 (pbpA) and penicillin-binding protein 5 (dacA) as well as a previously unidentified cell shape gene that we have called rodA. rodA mutants were spherical and very similar to pbpA mutants but were distinguishable from them in that they had no defects in the activity of penicillin-binding protein 2. The separation into two groups of spherical mutants with mutations that mapped close to lip was confirmed by complementation analysis. The genes dacA, rodA, and pbpA lie within a 12-kilobase region, and represent a cluster of genes involved in cell shape determination and peptidoglycan synthesis. A restriction map of the lip-leuS region was established, and restriction fragments were cloned from defective transducing phage into appropriate lambda vectors to generate plaque-forming phage that carried genes from this region. Analysis of the proteins synthesized from lambda transducing phage in ultraviolet light-irradiated cells of E. coli resulted in the identification of the leuS, pbpA, dacA, and lip gene products, but the product of the rodA gene was not identified. The nine proteins that were synthesized from the lip-leuS region accounted for 57% of its coding capacity. Phage derivatives were constructed that allowed about 50-fold amplification of the levels of penicillin-binding proteins 2 and 5 in the cytoplasmic membrane.

Amplification of a Mitochondrial DNA Sequence in the Cytoplasmically Inherited ‘Ragged’ Mutant of Aspergillus amstelodami

Abstract: A comparison has been made between mtDNA of the cytoplasmically inherited 'ragged' mutant of Aspergillus amstelodami and that of the wild-type strain. Ragged mitochondria contain both the wild-type mitochondrial genome and several large DNA molecules which are not cleaved by the restriction endonucleases BamHI, HaeIII, HhaI, HindII, HindIII, PstI and MboI, but are converted by either EcoRI or HpaII into a single 820-840 base-pair fragment. Restriction analysis and molecular hybridization data indicate that this fragment contains sequences of wild-type mtDNA located within a 1200-base-pair segment of the 40,500-base-pair genome, for which a basic restriction map has been deduced. It is concluded that in the ragged mutant a small segment of wild-type mtDNA has been amplified as tandem repeats, which is reminiscent of the Rho- petite phenotype of yeast. The results are discussed in relation to the phenomenon of senescence in Podospora anserina.

Intracisternal A-particle genes: identification in the genome of Mus musculus and comparison of multiple isolates from a mouse gene library.

Abstract: The genome of Mus musculus contains multiple copies (500 -1000) of DNA sequences related to the 35S RNA of intracisternal type A particles (IAPs). Using labeled IAP RNA as a probe in blot-hybridization experiments, we have identified a characteristic electrophoretic pattern of reactive fragments generated by restriction endonuclease cleavage of mouse DNA. From the genomic blots, we deduced a composite restriction map for a 6.5- to 7-kilobase (kb) DNA region containing sequences homologous to the IAP RNA. Units of this type appeared to be interspersed without obvious regularity in nonhomologous flanking regions. A 5.2-kb segment of this unit was inserted directly into plasmid pBR322 from HindIII/EcoRI digest of mouse DNA. The fragment was cloned and then labeled by nick-translation and used to scan a mouse embryo gene library (average 16-kb inserts in lambda Charon 4A); 1% of the library samples hybridized, confirming the extensive reiteration of IAP genetic units. Among six different library isolates containing 6.5- to 7-kb IAP units, some restriction sites were highly conserved whereas others varied in both occurrence and position. Despite this variation, heteroduplexes between the individual isolates showed continuous IAP homology regions of 7 kb. No flanking region homologies were seen in this limited sample. Some evidence suggests that mouse DNA may contain other dispersed sequence elements related to but smaller than the genetic unit defined above.

Restriction maps for twenty-one Charon vector phages.

Abstract: The mapping of the sites of cleavage of nine restriction endonucleases (EcoRI, HindIII, BamHI, SalI, KpnI, SstI, BglII, XhoI, and XbaI) on 21 Charon phage vectors is described. Maps of individual subsections were obtained and then combined to assemble the complete vector maps. Calculations of maximum and minimum sizes of inserts which may be carried by the vectors using different restriction endonucleases or pairs of restriction endonucleases are presented. The regions mapped include several parts of phi 80 that had not been mapped previously.

A general method for defining restriction enzyme cleavage and recognition sites.

Abstract: Publisher Summary This chapter discusses a general method for defining restriction enzyme cleavage and recognition sites. Class II restriction endonucleases cleave double-stranded DNA into specific fragments. These enzymes are powerful tools for the dissection of DNA; and they are essential for the construction of recombinant DNA molecules and for DNA sequence determination. These recognize specific sequences of nucleotides, four to six nucleotide pairs long. The wide variety in types of fragment termini has resulted in a number of strategies for characterizing the cleavage sites. These are based on the principle of 32 P-labeling the ends of a mixture of fragments produced by a given enzyme followed by determination of the nucleotide sequence at the labeled ends. The characterization of one cleavage site is sufficient to define a restriction enzyme recognition site, especially if the prediction is confirmed by the fragments produced using a DNA substrate whose sequence is completely known. In other cases, where the cleavage site is offset from the recognition site, a small number of sites must be compared.

Cloning and characterization of DNA sequences complementary to messenger ribonucleic acids coding for the synthesis of two surface antigens of Trypanosoma brucei

Abstract: Full length double-stranded complementary DNAs (ds cDNAs) could be synthesized on mRNAs enriched in sequences coding for the synthesis of the variant specific antigens (VSAs) AnTat 1.1 and AnTat 1.8 from Trypanosoma brucei brucei. The size of these ds cDNAs is about 1700 and 1850 base pairs for AnTat 1.1 and AnTat 1.8 respectively. The ds cDNAs were cloned in the plasmid pBR322; two clones harboring a copy of each coding sequence were selected. Both the hybrid-arrested translation and the positive hybridization elution methods confirmed that these recombinants contain VSA-specific inserts. A restriction map was constructed in each case. The two sequences seem to be inserted in a reversed 3'--5' orientation, respective to the plasmid polarity. The AnTat 1.8 cloned sequence is a palindrome probably due to a cloning artefact. Hybridization of the cloned DNAs with "Northern" blots of total or poly(A)+ RNA revealed in each case a single, specific band. The expression of these VSA genes appears thus to be regulated at the transcriptional level.

Mouse immunoglobulin genes: a bacterial plasmid containing the entire coding sequence for a pre-γ 2a heavy chain

Abstract: A DNA sequence complementary to the entire coding part of a mouse gamma 2a immunoglobulin heavy chain mRNA isolated from a myeloma producing a levan binding protein (UPC 10), has been cloned in the PstI site of pBR 322. Transformants containing sequences complementary to purified gamma 2a heavy chain mRNA were selected. One transformant, pG2a-10-21, containing a 1750 nucleotide insert, has been characterized by hybrid-arrested translation and purification of gamma 2a heavy chain mRNA on DNA-DBM cellulose filters. Restriction enzyme analysis and partial sequencing demonstrate that the pG2a-10-21 contains the complete structural sequence for the gamma 2a heavy chain and predicts the sequence of a 18 amino acid hydrophobic amino terminal extra piece segment.

Cloning and restriction mapping of the trmA gene coding for transfer ribonucleic acid (5-methyluridine)-methyltransferase in Escherichia coli K-12.

Abstract: A hybrid plasmid from the Clarke and Carbon collection has been isolated. This plasmid carries the trmA gene of E. coli, which is necessary for the formation of 5-methyluridine (m5U,ribothymidine) present in all transfer ribonucleic acid (tRNA) chains of the organism so far sequenced. A restriction map of the argCBH-trmA regions is presented. By using cloning in vitro, the trmA gene was located on a 2.9-kilobase pair deoxyribonucleic acid (DNA) fragment. These results and comparison with lambda dargECBH transducing phages established the gene order: argECBH trmA bfe in the 88-min region of the E. coli chromosomal map. Plasmids carrying this 2.9-kilobase pair DNA fragment overproduce the enzyme tRNA(m5U)methyltransferase (EC 2.1.1.35) 20 to 40 times. When this 2.9-kilobase pair chromosomal DNA fragment was expressed in a minicell system, a polypeptide of a molecular weight of 42,000 was synthesized. This polypeptide was tentatively identified as the tRNA(m5U)methyltransferase. These results support the earlier suggestion that the trmA gene is the structural gene for the tRNA(m5U)methyltransferase.

The nucleotide sequence and restriction enzyme sites of the polyoma genome.

Abstract: We present the 5295 nucleotide-long sequence of the polyoma genome and the restriction enzyme digestion sites predicted from this sequence.

Cloning and direct examination of a structurally abnormal human beta 0-thalassemia globin gene.

Abstract: Restriction endonuclease mapping permitted identification of a form of beta 0-thalassemia in which a partial deletion of the beta-globin structural gene occurred [Orkin, S H, Old, J M, Weatherall, D J & Nathan, D G (1979) Proc Natil Acad Sci USA 76, 2400-2404] To analyze its structure more directly, this abnormal human gene has now been cloned in bacteriophage lambda gtWES Restriction mapping of the cloned gene and of a normal beta-globin gene contained in the phage H beta G1 confirmed previous findings regarding the presence of a deletion toward the 3' end of the gene but could not establish its position more accurately Electron microscopy of the hybrid of the beta-thalassemia gene with globin RNA (R-loop analysis) provided unequivocal evidence for a deletion with the beta-globin structural gene Hybridization of restriction fragments of the mutant gene with homologous fragments of H beta G1 (heteroduplex analysis) revealed a continuous, internal deletion of about 06 kilobase of DNA in the mutant gene and permitted its localization within the beta-globin gene region This deletion removed the terminal third of the large intervening sequence within the beta-globin gene, the entire 3' coding block (extending from codon 105 to the end of the gene), and approximately 150 base pairs of DNA past the end of the normal globin gene

Unusual features in the nucleotide sequence of a cDNA clone derived from the common region of avian sarcoma virus messenger RNA

Abstract: We have constructed a recombinant plasmid containing a 700-base pair (bp) cDNA copy of the common region present at the 3' end of Schmidt-Ruppin avian sarcoma virus (ASV) 21S mRNA. The cDNA was inserted into plasmid pBR322 at the Pst I site by the G-C tailing method. A restriction map of the cloned insert from a recombinant plasmid pSRI indicates that it corresponds to the 3' end of the ASV genome. R-loop analysis with ASV genomic RNA indicates that the insert is colinear with the ASV genome over most of its length. The sequence of 331 bp at the 3' end of the DNA insert was determined and shows that the insert contains extra sequences not found at the 3' end of ASV genomic RNA. Following the terminally redundant sequence of 20 bp that has been found at the extreme 3' end of genomic RNA is a sequence of 79 bp that is almost identical to that located immediately next to the 20-bp repeat at the 5' end of ASV genomic RNA. This is followed by 18 bp of unique sequence, possibly of host origin. The structure of the clone suggests that ASV mRNA may differ from genomic RNA at its 3' end and that 21S mRNA is transcribed from integrated ASV DNA and contains at its 3' end sequences derived both from the 5' end of the ASV genome and from host DNA adjacent to the site of integration. The presence of termination codons in all three reading frames suggests that the common region probably does not contain coding sequences. However, the presence of sequences that resemble probable promoter sites supports the possibility that this region may be involved in the regulation of transcription.

Plasmids containing insertion elements are potential transposons.

Abstract: We studied in vivo recombination between the plasmid pHS1, a temperature-sensitive replication mutant carrying tetracycline resistance, and pSM1, a small plasmid carrying one copy of the insertion element IS1. Recombinant plasmids were found by selection for tetracycline resistance at 42 degrees C. Their formation was independent of recA function. Analysis of the physical structures of various recombinant DNA molecules with electron microscopy and restriction endonucleases revealed that pSMI was integrated at its IS1 into numerous sites on pHS1, giving rise to a duplication of IS1 in the same orientation at both junctions. Nucleotide sequence analysis of recombinant plasmids and their parental plasmid DNA revealed that nine nucleotides at a target site were duplicated at the junction of each IS1. This phenomenon implies that plasmids containing a translocatable DNA element can be potential transposons.