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At least 955 records · Page 53Linked to original sources
PubMed1999

An algorithmic approach to multiple complete digest mapping.

Multiple Complete Digest (MCD) mapping is a method of determining the locations of restriction sites along a target DNA molecule. The resulting restriction map has many potential applications in DNA sequencing and genetics. In this work, we present a heuristic algorithm for fragment identification, a key step in the process of constructing an MCD map. Given measurements of the restriction fragment sizes from one or more complete digestions of each clone in a clone library covering the molecule to be mapped, the algorithm identifies groups of restriction fragments on different clones that correspond to the same region of the target DNA. Once these groups are correctly determined the desired map can be constructed by solving a system of simple linear inequalities. We demonstrate the effectiveness of our algorithm on real data provided by the Genome Center at the University of Washington.

Algorithms↗
PubMed1990

The osmZ (bglY) gene encodes the DNA-binding protein H-NS (H1a), a component of the Escherichia coli K12 nucleoid.

A class of trans-acting mutations, which alter the osmoregulated expression of the Escherichia coli proU operon, maps at 27 min on the chromosome in a locus we have called osmZ. Mutations in osmZ are allelic to bglY, pilG and virR, affect gene expression, increase the frequency of the site-specific DNA inversion mediating fimbrial phase variation, stimulate the formation of deletions, and influence in vivo supercoiling of reporter plasmids. We have cloned the osmZ+ gene, mapped it at 1307 kb of the E. coli restriction map, identified its gene product as a 16 kDa protein, and determined the nucleotide sequence of the osmZ+ gene. The deduced amino acid sequence for OsmZ predicts a protein of 137 amino acid residues with a calculated molecular weight of 15,530. The primary sequence of OsmZ is identical to that of H-NS (H1a), a DNA-binding protein that affects DNA topology and is known to be associated with the bacterial nucleoid. Thus, osmZ is the structural gene for the H-NS (H1a) protein. The nucleotide sequence of osmZ is almost identical to that of hns; however, hns was incorrectly located at 6.1 min on the E. coli linkage map. Increased osmZ gene dosage leads to cell filament formation, altered gene expression, and reduced frequency of fimbrial phase variation. Our results suggest that the nucleoid-associated DNA-binding protein H-NS (H1a) plays a critical role in gene expression and in determining the structure of the genetic material.

Alleles↗
PubMed1985

Molecular cloning of integrated caprine arthritis-encephalitis virus.

A full-length DNA clone of the exogenous retrovirus, caprine arthritis-encephalitis virus (CAEV), was isolated from high molecular weight DNA of CAEV-infected Himalayan tahr ovary cells. Although other restriction maps of CAEV have been published, this is the first time that the proviral DNA has been cloned. The restriction enzyme map of the clone was determined and found to be identical to that of unintegrated linear CAEV DNA except for the presence of cellular flanking sequences. These findings establish that lentiviruses are able to integrate within the infected host cellular genome. The cloned CAEV genome was shown to contain terminal repeats of approximately 450 base pairs in length, and its restriction enzyme map was oriented with respect to the direction of viral RNA transcription. When the cloned CAEV DNA was used as a molecular probe, it failed to detect related proviral sequences in the genomes of a variety of vertebrate species, including the goat, sheep, horse, mouse, and man. When CAEV DNA was hybridized under relaxed conditions to a variety of cloned DNAs, representing different oncoviral genera, homology to mouse mammary tumor virus (MMTV) was observed, while no homology to avian type C or mammalian type A, C, and D retroviruses was detected. This homology was localized to a region in MMTV corresponding to the 3' end of the gag gene and the 5' end of the pol gene.

Animals↗
PubMed1989

Insertion of the SfMNPV polyhedrin gene into an AcMNPV polyhedrin deletion mutant during viral infection.

The Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin deletion mutant, d10A, was cotransfected with the recombinant plasmid SfMNPV.HindIII-L, which contains the Spodoptera frugiperda nuclear polyhedrosis virus (SfMNPV) polyhedrin gene. An occlusion-positive hybrid virus was obtained which contained a DNA insertion at approximately 13 map units on the AcMNPV.d10A genome. A fine structure restriction map of cloned viral DNA fragments from this region revealed that most of the 1.7-kb AcMNPV.PstI-O fragment (13.4-14.7 map units) had been replaced with a 3.7-kb fragment, containing the SfMNPV polyhedrin gene with flanking sequences and the entire pUC8 plasmid. Subclones of this region were sequenced and the exact site of insertion was determined. Flanking the insert were 43 and 60 bp of unknown origin at the 5' and 3' ends, respectively. Transcription was examined in the region of the insertion in both wild-type AcMNPV and the AcMNPV/SfMNPV (Ac/Sf) hybrid. In the AcMNPV, a nested set of seven overlapping transcripts ranging from 2.2 to 5.3 kb was found, each with coterminal 3' ends. Only the 2.2-kb transcript was found to be expressed early and throughout infection. The SfMNPV insertion interrupted this transcriptional unit and produced a more complex pattern of transcription. Alterations included a nested set of three overlapping transcripts with coterminal 5' ends, including the SfMNPV polyhedrin mRNA, transcripts originating in AcMNPV and terminating in either SfMNPV or pUC8 sequences, and other minor transcripts. The SfMNPV polyhedrin gene was sequenced and the locations of the 5' and 3' ends of polyhedrin mRNA were mapped. An analysis of SfMNPV polyhedrin protein expression showed that the SfMNPV polyhedrin gene in the hybrid virus was expressed at approximately one-fourth the level of the wild-type AcMNPV polyhedrin gene. Expression of a beta-galactosidase gene under the control of the SfMNPV polyhedrin promoter in the AcMNPV.d10A mutant was also investigated.

Amino Acid Sequence↗
PubMed1988

Escherichia coli gene purR encoding a repressor protein for purine nucleotide synthesis. Cloning, nucleotide sequence, and interaction with the purF operator.

The Escherichia coli gene purR, encoding a repressor protein, was cloned by complementation of a purR mutation. Gene purR on a multicopy plasmid repressed expression of purF and purF-lacZ and reduced the growth rate of host cells by limiting the rate of de novo purine nucleotide synthesis. The level of a 1.3-kilobase purR mRNA was higher in cells grown with excess adenine, suggesting that synthesis of the repressor may be regulated. The chromosomal locus of purR was mapped to coordinate 1755-kb on the E. coli restriction map (Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50, 495-508). Pur repressor bound specifically to purF operator DNA as determined by gel retardation and DNase I footprinting assays. The amino acid sequence of Pur repressor was derived from the nucleotide sequence. Pur repressor subunit contains 341 amino acids and has a calculated Mr of 38,179. Pur repressor is 31-35% identical with the galR and cytR repressors and 26% identical with the lacI repressor. These four repressors are likely homologous. Amino acid sequence similarity is greatest in an amino-terminal region presumed to contain a DNA-binding domain. A similarity is also noted in the operator sites for these repressors.

Amino Acid Sequence↗
PubMed1995

The erythromycin resistance gene of the Corynebacterium xerosis R-plasmid pTP10 also carrying chloramphenicol, kanamycin, and tetracycline resistances is capable of transposition in Corynebacterium glutamicum.

The clinical isolate Corynebacterium xerosis M82B carries the 50-kb R-plasmid pTP10 that confers resistance to the antibiotics chloramphenicol, kanamycin, erythromycin, and tetracycline. A detailed restriction map of pTP10 was constructed by cloning and analyzing restriction fragments of pTP10 in Escherichia coli. The resistance determinants of pTP10 were located by studying the phenotype of the recombinant plasmids in E. coli and Corynebacterium glutamicum. Restriction patterns of fragments encoding the kanamycin and erythromycin resistances revealed striking similarity to the kanamycin resistance of transposon Tn903 and the erythromycin resistance on plasmid pNG2 from Corynebacterium diphtheriae, respectively. Expression of the resistance determinants in E. coli and C. glutamicum ATCC 13032 led to high resistance levels in both strains, with the exception of the tetracycline resistance gene, which could be expressed only in C. glutamicum. Furthermore, the erythromycin resistance gene was found to be located on a transposable element which is functional in C. glutamicum strains.

Blotting, Southern↗
PubMed1992

Construction of a new shuttle vector for Lactobacillus.

To clone the malolactic enzyme gene from Lactobacillus sp. 89, construction of a shuttle vector able to express itself in Lactobacillus sp. 89 and Escherichia coli was undertaken. The shuttle plasmid pLE16 resulted from the union of pBR328 and of the pLB10 plasmid extracted from Lactobacillus bulgaricus 10. The bacterial transformation in Lactobacillus sp. 89 was performed by electroporation, and the clones were selected on MRS medium with 30 micrograms.mL-1 chloramphenicol added. Fifty percent of the clones from Lactobacillus sp. 89 lost their resistance to chloramphenicol following 28 generations when the selection pressure was not maintained. The restriction map of pLE16 (7600 bp) was established using several restriction enzymes.

Escherichia coli↗
PubMed1990

[Cloning of alpha-hemolysin gene from Staphylococcus aureus strain 015].

The genomic library of Staphylococcus aureus O15 has been constructed on the EMBL-3 vector. The synthetic oligonucleotide probes to N- and C-end regions of alpha-hemolysin permitted identification of the recombinant bacteriophage clone RS-1 containing a gene for this protein. The restriction map of the cloned fragment has been constructed for restriction endonucleases SalGI, EcoRV, PstI, PvuII. Expression of the alpha-hemolysin gene in phagolysate of the recombinant clone RS-1 (1000 units per ml) has been demonstrated.

Base Sequence↗
PubMed1989

[Cloning the genetic determinant of alpha-hemolysin and obtaining a series of insertional mutations in this determinant by the transposon Tn1000].

Functionally active genetic determinant of alpha-hemolysin was cloned. Hemolytic plasmid pHly195 was used as a donor of the determinant and pBR322 plasmid served as recipient. Cloning was done with a help of HindIII restriction endonuclease. The recombinant plasmid obtained represents pBR322 plasmid with the built-in fragment of 7.4 kb containing genes of functionally active determinant of alpha-hemolysin. Restriction map was constructed using HindIII, EcoRI, BamHI and SalI restriction endonucleases. Insertional mutagenesis was carried out with the help of the Tn1000 transposon. Plasmid DNAs were isolated from insertional mutants of Hly- phenotype and treated with EcoRI, SalI and BamHI. On the basis of the sizes of restriction fragments of the mutant plasmid DNAs localization and orientation of insertions of Tn1000 into the cloned determinant of alpha-hemolysin were determined.

Bacterial Proteins↗
PubMedDate not supplied

Cloning and organization of genes for 5S ribosomal RNA in the sea urchin. Lytechinus variegatus.

A 1.35-kb EcoRI fragment of Lytechinus variegatus DNA containing a single 5S rRNA gene has been cloned into the plasmid vector pACYC184. Four clones from different transformation experiments contain 5S rDNA inserts of about the same size and have the same restriction enzyme digestion patterns for the enzymes HaeIII, HinfI, HhaI, and AluI. One EcoRI site near the HindIII site of the plasmid vector pACYC184 is missing in all the four clones. By DNA sequencing, the missing EcoRI ws found to be EcoRI site, d(AAATTN)d(TTTAAN) in pLu103, one of the four 5S rDNA clones. The structure of pLu103 was determined by restriction mapping and blot hybridization. Three restriction fragments, 1.0-kb HaeIII/HaeIII, 0.375-kb AluI/AluI and 0.249-kb MboII/MboII, which contain the 5S rRNA coding region, have been subcloned into the EcoRI site of the plasmid pACYC184. The organization of 5S rRNA genes in the sea urchin genome was also investigated. It was found that restriction endonuclease HaeIII has a single recognition site within each 5S rDNA repeat, and yields two fragment lengths, 1.2 and 1.3 kb. The behavior of these 5S rRNA genes when total L. variegatus DNA is partially digested with HaeIII is consistent with an arrangement of 5S rRNA genes in at least two tandemly repeated, non-interspersed families. Both the coding region and spacer region of the 5S rRNA gene in pLu103 hybridize to 1.2 and 1.3-kb rDNA families. This indicates that the cloned EcoRI fragment of 5S rDNA in pLu103 represents one single repeat of 5S rDNA in the genome.

Animals↗
PubMed1993

Physical analysis of the terminal 240 kb of DNA from human chromosome 7q.

DNA from a 240-kb human telomeric yeast artificial chromosome (HTY) clone was analyzed using physical mapping methods. Cosmid subclones of the YAC were fingerprinted using restriction enzyme digestion and human repeat sequence hybridization and then assembled into two contigs that together span 93% of the human insert. Data from restriction mapping and Bal31 exonuclease experiments indicate that, except for the truncation of distal genomic (T2AG3)n sequences, the molecular clone HTY 146 contains a contiguous, 230-kb telomere-terminal fragment from 7qter. Markers derived from this clone will allow telomeric closure of the physical and genetic linkage maps of human chromosome 7q.

Base Sequence↗
PubMed1988

Close physical linkage of the murine Ren-1 and Ren-2 loci.

In addition to the Ren-1 gene common to all mice, some inbred strains carry a second copy of the renin structural gene, Ren-2. These two loci are tightly linked genetically on mouse chromosome one. We have used pulsed field gel electrophoresis (PFGE) to study the physical arrangement of the two renin genes in the inbred strain DBA/2. PFGE mapping permitted the construction of a restriction map of the Ren loci spanning roughly 120 Kb. The results indicate that the genes are transcribed in the same relative direction, that Ren-2 lies upstream relative to Ren-1, and that the respective coding sequences are separated by approximately 20 Kb.

Animals↗
PubMed1989

Complete maps of IS1, IS2, IS3, IS4, IS5, IS30 and IS150 locations in Escherichia coli K12.

In this paper complete distribution maps are presented of the seven IS elements 1, 2, 3, 4, 5, 30 and 150. These maps were obtained during the construction of an almost complete restriction map of the Escherichia coli genome of K12 strain BHB2600. The positions of IS elements were correlated to this map. The distribution of integration sites of all IS types is nonrandom. Besides a large gap from 79 min to 96 min, there is a pronounced IS cluster at 6 min and another at 97 min, map locations that have low gene incidences on the classical map. One cluster coincides with a region of IS induced rearrangements. The IS distribution pattern was compared to patterns of strains W3110 and HB101.

Cosmids↗
PubMed1992

Equine herpesvirus 5: comparisons with EHV2 (equine cytomegalovirus), cloning, and mapping of a new equine herpesvirus with a novel genome structure.

A new equine herpesvirus, provisionally designated equine herpesvirus 5 (EHV5; Browning and Studdert (1987) J. Gen. Virol. 68, 1441-1447), was examined for the degree of genomic difference from equine herpesvirus 2 (EHV2) by Southern hybridizations. EHV5 and EHV2 whole genomic DNA probes were highly specific for homologous DNA only, indicating that significant genomic difference exists between the two viruses. Restriction endonuclease analysis of EHV5 strain 2-141 (EHV5.2-141) revealed that the genome is 179 kb and exists as a single isomer. Clones representing 82% of the genome were obtained and used to construct restriction maps for four restriction endonucleases. Hybridization experiments indicated that the EHV5.2-141 genome does not contain large terminal or internal repeats, although some evidence for very short repeated sequences in the genomic termini was obtained. Such a genome structure makes EHV5 unique among the equine herpesviruses but similar to the mouse, rat, and guinea pig cytomegaloviruses and the tupaiid herpesvirus. Sequence analysis of one of the genomic termini of EHV5.2-141 revealed the presence of a 30-bp sequence (pac-1; Deiss et al. (1986) J. Virol. 59, 605-618) which is highly conserved among herpesviruses.

Animals↗
PubMed1991

Detailed physical map and set of overlapping clones covering the genome of the archaebacterium Haloferax volcanii DS2.

An integrated approach of "bottom up" and "top down" mapping has produced a minimal set of overlapping cosmid clones covering 96% of the 4140 kilobase-pairs (kbp) Haloferax volcanii DS2 genome and a completely closed physical map. This genome is partitioned into five replicons: a 2920 kbp chromosome and four plasmids, of 690 kbp (pHV4), 442 kbp (pHV3), 86 kbp(pHV1) and 6.4 kbp (pHV2). A restriction map for six infrequently-cutting restriction enzymes was constructed, representing a total of 903 sites in the cloned DNA. We have placed the two ribosomal RNA operons, the genes for 7 S RNA and for RNaseP RNA and 22 protein-coding genes on the map. Restriction site frequencies show significant variation in different portions of the genome. The regions of high site density correspond to halobacterial satellite or FII DNA which includes two small regions of the chromosome, the plasmids pHV1 and pHV2, and half of pHV4, but not pHV3.

Archaea↗
PubMed1989

Localization of the Escherichia coli rnt gene encoding RNase T by using a combination of physical and genetic mapping.

The rnt gene encoding RNase T was cloned on a 13-kilobase BamHI fragment. Restriction analysis of the fragment and comparison of it with the Escherichia coli restriction map localized rnt to kilobase coordinates 1733 to 1746, corresponding to about 36 min on the genetic map. The map location was confirmed by cotransduction with the nearby zdg-229::Tn10 and ksgB1 markers.

Escherichia coli↗
PubMed2003

Intra-specific and inter-specific recombination of tortricid-specific granuloviruses during co-infection in insect larvae.

Intra-specific recombination between two genotypes of the Cryptophlebia leucotreta granulovirus (CrleGV), namely CV3 and CV4, was studied by mixed infection experiments of larvae of C. leucotreta, followed by in vivo cloning and DNA restriction enzyme analyses of isolated progeny viruses. As a prerequisite for these studies a comparative restriction map for of CV3 and CV4 was constructed for eight restriction enzymes. The mixed infection experiments resulted in the isolation of the recombinant CrleGV CVR, which contained restriction sites typical for both parental viruses. Inter-specific recombination between two different granulovirus species, namely CrleGV CV3 and Cydia pomonella granulovirus (CpGV), was analogously investigated by mixed infections of larvae of C. leucotreta. A survey of more than 300 isolated CrleGV and CpGV clones did not reveal any recombinant, which indicated an extremely low recombination frequency in these experiments. By using a specific PCR approach, however, chimerical fragments from the highly conserved granulin gene sequence could be observed in DNA preparations of virus progeny. Cloning and sequencing indicated recombination between CrleGV and CpGV DNA. Our results suggest that recombination between granulovirus genotypes and granulovirus species result in eventually viable viruses and may contribute to the genetic diversity in this virus group.

Animals↗