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PubMed1985

Characterization of restriction endonuclease maps of hepatitis B viral DNAs.

The HBV DNA isolated from Dane particles of 9 patients' plasma was cloned into the EcoRI or BamHI site of the pUC8 plasmids. Two plasmids with full length HBV DNA and four plasmids containing the HBV surface antigen gene were obtained. Based on our cloned HBV DNA and a comparison with 7 complete sequences and 5 restriction endonuclease patterns of HBV DNA published by others, we can recognize common restriction sites shared by different subtypes (adw, adr, ayw, and adyw): (1) a HincII site in the S gene, (2) a BamHI site in the X region, and (3) two BglII sites in the C gene. In addition adw has specific sites for HincII, BamHI, and PstI in the pre-S region. A unique XhoI site is present in the pre-S region in all subtypes except for adw.

Bacterial Proteins↗
PubMed1984

Isolation of the Candida albicans gene for orotidine-5'-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations.

A gene bank of Sau3A partially digested Candida albicans DNA in vector YEp13 was used to complement a ura3 mutation (orotidine-5'-phosphate decarboxylase, OMPdecase) in S. cerevisiae. Two plasmids which complemented ura3 and showed clear linkage of Ura+ and plasmid markers were selected for further study. Both plasmids also complemented the corresponding OMPdecase mutation (pyrF) in E. coli. Restriction mapping and subcloning studies localized the OMPdecase complementing activity to a region common to both plasmids. Probes prepared from this common region hybridized specifically to C. albicans DNA and not to E. coli or S. cerevisiae DNA. Southern blot analysis also showed that the restriction map of the ura3 complementing region of one plasmid was colinear with C. albicans genomic DNA. Expression of the OMPdecase complementing gene in E. coli and S. cerevisiae was not dependent upon orientation relative to vector sequences, suggesting that promotion could be occurring within the C. albicans fragment. Expression was sufficient to allow complementation in S. cerevisiae with integrating as well as high copy number vectors.

Candida albicans↗
PubMed1992

Physical maps of the genomes of three Bacillus cereus strains.

NotI restriction maps of the chromosomes from Bacillus cereus ATCC 10876, ATCC 11778, and the B. cereus type strain ATCC 14579 have been established and compared with the previously established map of B. cereus ATCC 10987. Between 10 and 14 NotI fragments were observed, ranging from 15 to 1,300 kb, in digests of DNA from the various strains. The sizes of the genomes varied between 5.4 and 6.3 Mb. The maps were constructed by hybridization of 42 random probes, prepared from B. cereus ATCC 10987 libraries, to fragments from partial and complete NotI digests, separated by pulsed-field gel electrophoresis. Nine probes were specific for ATCC 10987 only. Probes for five B. subtilis and five B. cereus genes were also used. The NotI restriction fragment patterns of the four strains were strikingly different.

Bacillus cereus↗
PubMed1978

Anatomy of bovine mammillitis DNA. I Restriction endonuclease maps of four populations of molecules that differ in the relative orientation of their long and short components.

In this paper, we report that the DNA of bovine mammillitis virus (BMV) consists of two covalently linked components that are 71.5 x 10(6) and 15.7 x 10(6) in molecular weight and designated L and S, respectively. We further report that the BMV DNA consists of four equimolar populations differing only in the orientation of the L and S components relative to each other. This conclusion is based on the following: (i) The sum molecular weight of fragments generated by digestion of BMV DNA with Hsu I, Hpa I, Bgl II, or Xba I significantly exceeds the established molecular weight of the intact DNA. (ii) In each digest, the fragments form three groups differing in molar concentration. In reference to the molar concentration of intact DNA, each enzyme digest contained a set of four fragments 0.25 M in concentration, a set of four fragments 0.5 M in concentration, and a variable size set, unique for each enzyme digest, 1.0 M in concentration. (iii) Experiments involving digestion of intact DNA by lambda exonuclease followed by restriction endonuclease digestion established that each of four 0.5 M fragments were positioned at the termini of the BMV DNA. (iv) Complete maps for the fragments generated by each enzyme established that the 0.25 M fragments arise by fusion of the sequences of the terminal fragments when these are juxtaposed as a consequence of the inversion of L and S components. The maps also established the dimensions of the L and S components. We conclude that the structure of BMV DNA is similar to that of HSV DNA previously shown to consist of two unequal size components that invert relative to each other.

Base Sequence↗
PubMed1993

In situ hybridization mapping and restriction fragment length polymorphism analysis of the porcine albumin (ALB) and transferrin (TF) genes.

In situ hybridization analyses were conducted on porcine metaphase chromosomes using porcine liver albumin (ALB) and transferrin (TF) cDNA probes. The ALB gene was assigned to the q12 band of chromosome 8 and the TF gene to the q31 band of chromosome 13. For the latter, a statistically significant secondary peak was observed on the 6p15 band. However, the TF probe predominantly hybridized to the 13q31 band, indicating that this band is the most likely site of the TF gene. Since the TF gene belongs to linkage group V, this linkage group can now be assigned to chromosome 13. The TF and ALB probes were also used for restriction fragment length polymorphism (RFLP) analysis. A screening of 10 unrelated animals revealed TaqI RFLPs for both ALB and TF. Family studies indicated that the ALB and TF polymorphisms were controlled by three and two alleles, respectively.

Alleles↗
PubMed1996

Analysis of the erythropoietin receptor gene in patients with myeloproliferative and myelodysplastic syndromes.

The human erythropoietin receptor (EpoR) gene has been cloned and characterized. Very few EpoR genetic abnormalities have been reported so far. Polycythemia vera (PV) is characterized by low/normal serum erythropoietin (Epo) levels with proposed Epo hypersensitivity. Myelodysplastic syndromes (MDS) are characterized by refractory anemia with variable serum Epo levels. Several reports have suggested EpoR abnormalities in both types of stem cell disorders. We analyzed DNA obtained from peripheral blood mononuclear cells of seven healthy controls, 20 patients with myeloproliferative disorders (MPD, 11 patients with PV, five agnogenic myeloid metaplasia with myelofibrosis, four essential thrombocytosis) and eight patients with refractory anemia with ringed sideroblasts (RARS), an MDS variant. The DNA was digested with four restriction enzymes (BamHI, Bgl II, Sacl and HindIII), followed by Southern blot, using a 32P radiolabeled probe, containing 1.5 kb of the human EpoR cDNA. All 20 MPD patients and seven out of the eight MDS patients demonstrated a restriction pattern which was identical to the seven normal controls, as well as to the erythroid cell line K562, and also consistent with the expected restriction map, for all four enzymes tested. One RARS patient had a normal pattern with three enzymes but a different one with HindIII. The HindIII 12 kb large band was replaced by a faint 12 kb band and a new (about 9 kb) band appeared. The EpoR restriction map and the normal pattern obtained with the other three enzymes suggest that this patient has a 3 kb upstream deletion in one allelic EpoR gene. The same molecular pattern was detected in the patient's sister, who suffers from anemia with mild bone marrow (BM) dyserythropoiesis and plasmacytosis. Northern blot analysis showed that the patient's BM RNA carried normal EpoR message. This familial pattern may represent polymorphism. However, the patient's very high serum Epo level, her resistance to treatment with recombinant Epo, and the abnormally low growth rate of in vitro erythroid cultures, suggesting poor response to Epo in this MDS patient as well as the hematological abnormalities in her sister, support the speculation that the different EpoR gene might serve as a genetic predisposing marker and potentially could be involved (probably via post-transcriptional mechanisms and by an interaction with other factors or cytokines) in the pathogenesis. Our data suggest that the EpoR is intact in MPD and in most patients with RARS. One RARS patient had a familial different genetic structure, which could represent polymorphism. However, we can speculate also that it might be involved in the pathogenesis of the disease.

Aged↗
PubMed1986

Inducible gene expression by DNA rearrangements in human cells.

A permanent human cell line, cell line LM205, was established by transforming primary human fibroblasts with a plasmid containing both simian virus 40 sequences with a defective origin of replication and a G418 resistance gene (neo) that lacked a eucaryotic transcriptional promoter. G418-resistant cells appeared spontaneously in clonal populations of LM205 cells at a frequency of approximately 10(-5) cell per cell plated in the presence of 400 micrograms of G418 per ml. G418 resistance was stable and correlated with the appearance of neo-specific RNA. Characterization of the neo gene in the G418-sensitive parental cell line by both a Southern blot analysis and a restriction map analysis of cloned sequences demonstrated that there was a stable integration site containing a single neo coding sequence. A Southern blot analysis of five G418-resistant subclones indicated that there were heterogeneous DNA rearrangements in the region of the neo gene that were unique in each subclone. Restriction mapping of a fragment containing the neo gene isolated from one of the resistant subclones demonstrated that the rearrangement was a tandem duplication that resulted in the relocation of the simian virus 40 bidirectional transcriptional promoter 5' to the neo gene. Tandem duplication was also consistent with the Southern blot polymorphisms observed in the other resistant subclones, suggesting that there were heterogeneous sites of recombination with respect to both the neo gene and the simian virus 40 promoter. Although these rearrangements resulted in an increase in neo gene copy number per cell, amplification showed no correlation quantitatively with the large increase in neo-specific RNA in these cells. Therefore, G418-resistant colony formation in cell line LM205 provides a method for studying both the mechanisms involved in this type of recombination and the factors influencing its frequency.

Cell Line↗
PubMed1982

Molecular cloning of murine endogenous viral sequences and expression of a newly constructed recombinant murine leukemia virus DNA in transfected mink cells.

In the process of molecularly cloning unintegrated proviral DNA from NIH-3T3 mouse cells infected with Rauscher murine leukemia virus, we observed the occurrence of clones with inserts smaller than the expected Rauscher murine leukemia virus fragments. The insert of one of these clones, lambda.Xe-1, was characterized in more detail. It had a size of 3.5 kilobases. The restriction map was similar but not identical to that of the envelope regions of Moloney and Rauscher murine leukemia viruses. After ligation to previously cloned Moloney murine leukemia viral sequences and transfection of the ligated DNA into mink lung cells a nondefective xenotropic murine leukemia virus, XH-19, was isolated. Restriction mapping of proviral DNA isolated from mink lungs cells chronically infected with XH-19 showed the presence of Moloney murine leukemia virus-derived sequences coupled to xenotropic viral sequences.

Animals↗
PubMed1987

Molecular characterization of the Akvr-1 restriction gene: a defective endogenous retrovirus-borne gene identical to Fv-4r.

A dominant restriction allele, Akvr-1r, from California wild mice (Mus musculus domesticus) confers resistance to exogenous ecotropic murine leukemia virus (MuLV) infection. The presence of an ecotropic MuLV envelope-related glycoprotein in uninfected virus-resistant cells suggests that viral interference is a possible mechanism for this resistance. We molecularly cloned the ecotropic MuLV envelope-related sequence from the genomic DNA of a wild mouse homozygous for the Akvr-1r locus. The cloned provirus was defective and contained a C-terminal end of the pol gene, a complete envelope gene, and a 3' long terminal repeat. The presence of this provirus was directly correlated with Akvr-1r-mediated virus resistance in cell cultures and hybrid mice. The Akvr-1r provirus restriction map and partial DNA sequence were identical to those of the Fv-4r allele, an ecotropic MuLV resistance locus from Japanese feral mice (M. musculus molossinus), which was previously shown to be allelic with the Akvr-1r gene. The 3' host flanking sequences of Fv-4r and Akvr-1r also had identical restriction maps. These findings indicate that Akvr-1r and Fv-4r are the same gene. It was probably acquired by interbreeding of these feral species in recent times. Conservation of this locus might be favored by the useful function that it performs in protection against ecotropic MuLV infection endemic in both populations of wild mice.

Alleles↗
PubMed1986

Molecular cloning and characterization of sequences from the regulatory cluster of the Pseudomonas plasmid alk system.

Alkane oxidation functions encoded by the Pseudomonas plasmid CAM-OCT are positively regulated by one or more products of a locus designated alkR. To characterize this locus in greater detail, molecular cloning and restriction mapping of sequences covering the alkR region have been carried out in Escherichia coli, followed by mobilization to Pseudomonas recipients for analysis of genetic content. Inserts from Pseudomonas (CAM-OCT) strains were cloned into vectors pLAFR1, the pLAFR1::Tn7S derivative pXJS5403, and the transposon vector Tn3 delta 596. This has made it possible to: (1) construct a detailed restriction map of cloned fragments and the alkR region of CAM-OCT; (2) map insertion sites of the transposon Tn7S into alkR cistrons; and (3) analyze the ability of cloned sequences to complement or effect marker rescue of alkR nitrosoguanidine- and Tn7S-induced mutations. In addition, transcription of an alkB'-lacZ transcription fusion in the presence of a cloned 18.5 kb EcoRI alkR fragment and an inducer of the alk system confirmed that our cloned sequences contain functional alkR cistrons. The complementation/marker rescue results indicate that alkR is a complex locus and that the products of at least three cistrons are required for the complete AlkR+ phenotype. One of these cistrons is identified by mutations which alter a component of the inducer recognition system.

Alkanes↗
PubMed1991

Physical and genetic map of the Methanococcus voltae chromosome.

A physical map of the Methanococcus voltae chromosome was constructed on the basis of restriction mapping and cross-hybridization experiments, employing total and partial digests obtained with rarely cutting restriction enzymes. On the basis of the sum of the fragment sizes of digests with seven enzymes the chromosome length was calculated to be approximately 1900 kb. The derived map is circular. Hybridization of gene probes to mapped restriction fragments has led to a genetic map of genes for structural RNAs as well as proteins, including enzymes involved in the methanogenic pathway.

Archaea↗
PubMed2003

Genomic EcoRI polymorphism and cosmid sequencing reveal an insertion/deletion and a new IGLV5 allele in the human immunoglobulin lambda variable locus (22q11.2/IGLV).

The human immunoglobulin lambda locus ( IGL) is mapped at Chromosome 22q11.2, spanning about 1 Mb of DNA, and directs the synthesis of lambda-type immunoglobulin light chains. The positions of about 73-74 germline V-lambda genes, depending on the haplotypes, are known, with 29-33 of them being functional IGLV genes. These genes were divided into 11 subgroups ( IGLV1 to IGLV11) distributed into three gene clusters ( VA, VB, and VC). We constructed a high-resolution restriction map of a 37-kb cosmid clone (cosmid 8.3) harboring genes of the IGLV1, IGLV7, and IGLV5 families and the non-coding sequences IGLV(I)-42 and IGLV(VII)-41-1, located at cluster VB of the IGL locus. These IGLV genes were associated with unique EcoRI fragments detectable in Southern blots of genomic DNA. Population RFLP has revealed new IGLV alleles and haplotypes. We used the restriction map of cosmid 8.3 and the IMGT database as a reference for RFLP studies. EcoRI Southern blot hybridizations with subgroup-specific probes of the functional and open reading frame sequences present in cosmid 8.3 revealed different frequencies of IGLV gene fragments, as well as deletions of IGLV1-50 and IGLV5-39 genes and RFLP involving IGLV5-45 and IGLV5-48 genes. All members of the IGLV7 subgroup were monomorphic. Sequencing of the genes present in cosmid 8.3 revealed a new allelic variant of the IGLV5 subgroup. These data contribute to a better understanding of the contribution of the germline IGLV genes to the human genetic background and polymorphism.

Alleles↗
PubMed1983

Molecular cloning of bovine leukemia virus DNA integrated into the bovine tumor cell genome.

The bovine leukemia virus (BLV) DNA harbored in the bovine tumor cell genome was cloned in lambda Charon 4A phage. Using either representative or 3' half-enriched BLV cDNA as a blot hybridization probe, clone lambda BLV-1 was shown to carry 9 kb of the BLV genome, flanked by cellular sequences at both ends. Restriction mapping with twelve endonucleases and hybridization of the DNA fragments to BLV cDNA representing a 3'-end portion of the viral genome revealed the presence and precise location of two long terminal repeats (LTRs) and virus-cell junctions. Thus, lambda BLV-1 appears to contain the complete BLV genome and flanking tumor cellular sequences. The restriction map of the cloned BLV proviral DNA closely resembles that previously reported for unintegrated linear proviral DNA, but differs significantly from that of the integrated provirus of another BLV isolate, the difference occurring preferentially in the putative gag and pol genes.

Animals↗
PubMedDate not supplied

[Isolation and molecular-genetic characteristic of a cryptic plasmid from the Francisella novicida like F6168 strain].

A 4 Kb plasmid DNA has been isolated from Francisella novicida like strain F6168. Restriction map of the plasmid was constructed for restriction endonucleases HindIII, XbaI, EcoRV, BgIII. The plasmid pFN10 has been shown to be stably inherited by F. tularensis. The use of pFN10 for the construction of plasmid vectors for microorganisms of the genus Francisella is discussed.

DNA, Recombinant↗
PubMed1985

Aerobactin genes in clinical isolates of Escherichia coli.

The location of the aerobactin gene complex on either the chromosome or plasmid was determined in eight aerobactin-positive clinical isolates of Escherichia coli by Southern hybridization analysis, using as probes the cloned aerobactin genes from the ColV-K30 plasmid. The aerobactin genes were in two cases detected on large plasmids, whereas in the other strains the aerobactin genes are most likely located on the chromosome. Restriction mapping revealed only slight variations in the structural genes and an at least 3.4-kilobase-long upstream region conserved in all three plasmid-coded systems. A 7.7-kilobase HindIII fragment upstream and adjacent to the 16.3-kilobase HindIII fragment carrying the complete aerobactin system was cloned from the ColV-K30 plasmid. Fine-structure restriction mapping identified the left insertion sequence in the upstream region as IS1, in inverted orientation to the IS1 element downstream from the aerobactin operon. The upstream and downstream sequences of IS1 appear to have perfect homology, as indicated by S1 nuclease resistance of a 760-base-pair DNA duplex formed by both IS1 elements.

Base Sequence↗
PubMed1991

Polymorphism of Nopaline-type T-DNAs from Agrobacterium tumefaciens.

The structure of several T-DNAs of Agrobacterium tumefaciens was determined by molecular cloning and Southern hybridization. The T-DNAs cloned in Escherichia coli vectors from four different nopaline type strains (PyTE1, PO31, PO22, and AKE10) showed various sizes of restriction enzyme fragments. Comparative analysis of the restriction maps revealed that the T-DNAs were composed of three distinct structural domains: (1) the region proximal to the right border (Domain I) containing the portion essential for tumorigenicity, (2) the proximity to the left border (Domain II), and (3) the region between the two domains (Domain III) to both of which no functional assignments have yet been made. The restriction map indicated that the Domains I and II were conserved in the most clones, including the well-characterized T37 T-DNA. The only exception was AKK1 (obtained from AKE10) which differed in Domain I. In the Domain III, insertions of 1.5- or 1.6-kb DNA were found in four clones, whereas an additional 2.5-kb insertion was found in one clone (PO22P1). The individual T-DNAs including Domain III with insertions was demonstrated in petunia and poplar tumors induced by the referred A. tumefaciens strains. However, resulting tumors differed in morphology and growth. These results suggest that the length polymorphism of the nopaline type T-DNA can be accounted by DNA insertions, and that diverse T-DNAs reflect their different roles in tumorigenicity.

Arginine↗
PubMed1983

Analysis of four tobacco mitochondrial DNA size classes.

Supercoiled mtDNAs were isolated from tobacco suspension culture cells and three of the smallest size classes (10.1, 20.2 and 30.3 kb) were characterized through denaturation, heteroduplex and restriction mapping. The 20.2 molecule was found to be a head-to-tail dimer of the 10.1 or X size class, while the 30.3 kb size class was found to contain two kinds of molecules, a head-to-tail trimer of X (X3) and a second molecule, ABC. X and ABC had a 118 +/- 35 bp region of homology, and both size classes shared a degree of homology with at least one other size class. Restriction maps of both the X and ABC molecules are presented and the possible origin and role of the many plant mtDNA size classes are discussed.

Base Composition↗