Showing papers on "Restriction map published in 1991"

Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast.

Abstract: The methods described in this chapter permit the manipulation of virtually any cloned yeast chromosomal sequence by virtue of the fact that DNA transformed into yeast integrates into the chromosome by homologous recombination. Furthermore, double-strand breaks in transforming DNA stimulate recombination and can be used to target integration events. This allows simple one-step gene disruption methods using yeast selectable markers. The availability of counterselectable markers makes it possible to replace chromosomal sequences with mutant alleles that cannot be directly selected. Finally, these same methods can be used to rescue chromosomal alleles on plasmids for subsequent molecular analysis.

Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes.

Abstract: DNA sequences from specific genes, amplified by the polymerase chain reaction technique, were used as substrata for nonisotopic restriction endonuclease fragment length polymorphism differentiation of rickettsial species and genotypes. The products amplified using a single pair of oligonucleotide primers (derived from a rickettsial citrate synthase gene sequence) and cleaved with restriction endonucleases were used to differentiate almost all recognized species of rickettsiae. A second set of primers was used for differentiation of all recognized species of closely related spotted fever group rickettsiae. The procedure circumvents many technical obstacles previously associated with identification of rickettsial species. Multiple amplified DNA digest patterns were used to estimate the intraspecies nucleotide sequence divergence for the genes coding for rickettsial citrate synthase and a large antigen-coding gene of the spotted fever group rickettsiae. The estimated relationships deduced from these genotypic data correlate reasonably well with established rickettsial taxonomic schemes.

Analysis of the physical state of different human papillomavirus DNAs in intraepithelial and invasive cervical neoplasm.

Abstract: The integration of human papillomavirus (HPV) DNA into the human genome has been generally accepted as a characteristic of malignant lesions. To gain a better understanding of this phenomenon, genomic DNA from 181 cervical biopsy specimens was isolated and analyzed for HPV type and physical state of the HPV genome. These specimens represented the full spectrum of cervical disease, from condyloma to invasive carcinoma. Discrimination between integrated and episomal HPV DNA was accomplished by the detection of HPV-human DNA junction fragments on Southern blots. In most cases in which ambiguous Southern blot results were obtained, the specimens were reanalyzed by two-dimensional gel electrophoresis. Of the 100 biopsy specimens of cervical intraepithelial neoplasia analyzed, only 3 showed integrated HPV DNA, in contrast to 56 (81%) of 69 cervical carcinomas (P less than 0.001) showing integrated HPV DNA. Of the 40 carcinomas containing HPV 16 DNA, 29 (72%) had integrated HPV DNA, of which 8 (20%) also had episomal HPV DNA. In 11 (27%) cancers, only episomal HPV 16 DNA was detected. All 23 HPV 18-containing carcinomas had integrated HPV DNA, and 1 also had episomal HPV 18 DNA. The difference between HPV types 16 and 18 with respect to frequency of integration was statistically significant (P less than 0.01). The results of this study indicate that detectable integration of HPV DNA, regardless of type, occurs infrequently in cervical intraepithelial neoplasia. The absence of HPV 16 DNA integration in some carcinomas implies that integration is not always required for malignant progression. In contrast, the consistent integration of HPV 18 DNA in all cervical cancers examined may be related to its greater transforming efficiency in vitro and its reported clinical association with more aggressive cervical cancers.

Polymorphic structure of the tumor necrosis factor (TNF) locus: an NcoI polymorphism in the first intron of the human TNF-beta gene correlates with a variant amino acid in position 26 and a reduced level of TNF-beta production.

Abstract: Since a dysregulated synthesis of tumor necrosis factor alpha (TNF-alpha) may be involved in the pathogenesis of autoimmune diseases, it was of interest to precisely locate the recently reported NcoI restriction fragment length polymorphism (RFLP) of the TNF-alpha region. However, by mapping of 56.8 kb of overlapping cosmid clones and direct sequencing, we could localize the polymorphic NcoI restriction site within the first intron of the TNF-beta gene and not in the TNF-alpha gene. To study whether regulatory mechanisms are affected by this polymorphism, we analyzed the TNF-alpha/TNF-beta production of phytohemagglutinin-stimulated peripheral blood mononuclear cells of individuals homozygous for the TNF-beta NcoI RFLP by ELISA and concomitant Northern blot analysis. On days 2-4 after stimulation with mitogen, the TNFB*1 allele corresponding to a 5.3-kb NcoI fragment presented with a significantly higher TNF-beta response. A mRNA analysis demonstrated that higher protein levels of TNF-beta correlate also with increased amounts of TNF-beta transcripts. No allelic association was found in respect to TNF-alpha production. To further investigate a possible allelic influence on transcription, we determined the DNA sequence of 2 kb of the 5' portion of our cloned TNFB*2 allele and compared it with the available TNF-beta sequences. By computer-aided recognition motif search of DNA binding factors, we report putative binding sites conserved between mouse and man in the 5' flanking region as well as in intron 1 of the TNF-beta gene, found also in other cytokine promoter sequences. In addition, by polymerase chain reaction amplification and sequencing of 740 bp of the 5' part of TNF-beta of individuals typed homozygously for the NcoI RFLP, we could show that amino acid position 26 is conserved as asparagine in the TNFB*1 and as threonine in the TNFB*2 sequence. A previously reported, EcoRI RFLP in the 3' untranslated region of TNF-beta does not segregate with either of the two alleles. Thus, four TNFB alleles can be defined at the DNA level.

A rapid procedure for extracting genomic DNA from leukocytes

Abstract: 1. Collect 5 ml of blood in a vacutainer tube (Becton Dickinson) containing EDTA and mix. 2. Make volume up to 10 ml with solution 1 (10 mM Tris pH 7.6; 10 mM KC1; 10 mM MgCy. 3. Add 120 /il Nonidet P40 (BDH) to lyse the cells. Mix well by inverting several times. 4. Spin down the nuclear pellet at 2000 rpm for 10 mins. 5. Pour off the supernatant without dislodging the pellet. The pellets can be stored frozen. 6. Gently resuspend pellet well in 800 /il of solution 2 (10 mM Tris pH 7.6; 10 mM KC1; 10 mM MgCl2; 0.5 M NaCl; 0.5% SDS; 2 mM EDTA). Solution 2 will lyse the nuclei so be careful not to shear the DNA. Transfer to a 1.5 ml microcentrifuge tube. 7. Add 400 /il of distilled phenol (saturated with 1 M Tris pH 8.0) and mix well. 8. Microfuge for 1 min at 12000 rpm. Transfer upper phase to a clean microfuge tube. Do not worry about transferring a little of the interface. 9. Add 200 /tl of phenol and 200 /il of chloroform :isoamyl alcohol (24:1). Mix well by inverting. 10. Spin for 1 min at 12000 rpm. Transfer upper phase to a clean microfuge tube. 11. Add 700 /tl of chloroform:isoamyl alcohol and extract as above. 12. Transfer upper aqueous phase to a small clean container. Avoid removing the interface. Add 2 volumes of ice cold ethanol and mix to precipitate the DNA*. 13. With the sealed tip of a pasture pipette, transfer the DNA fibres to a microcentrifuge tube containing 1 ml of 70% ethanol. Mix well to wash the DNA. 14. Spin for 5 mins at full speed. Discard the ethanol and dry pellet in a speed vac. Resuspend DNA in sterile water at 65°C. Do not over-dry genomic DNA or it will be difficult to resuspend. SIZE A / /Kb Hindll l

A genomic scanning method for higher organisms using restriction sites as landmarks.

Abstract: We have developed a powerful genomic scanning method, termed "restriction landmark genomic scanning," that is useful for analysis of the genomic DNA of higher organisms using restriction sites as landmarks. Genomic DNA is radioactively labeled at cleavage sites specific for a rare cleaving restriction enzyme and then size-fractionated in one dimension. The fractionated DNA is further digested with another more frequently occurring enzyme and separated in the second dimension. This procedure gives a two-dimensional pattern with thousands of scattered spots corresponding to sites for the first enzyme, indicating that the genome of mammals can be scanned at approximately 1-megabase intervals. The position and intensity of a spot reflect its locus and the copy number of the corresponding restriction site, respectively, based on the nature of the end-labeling system. Therefore, this method is widely applicable to genome mapping or detection of alterations in a genome.

Analysis of Interstrain Variation in Cytomegalovirus Glycoprotein B Sequences Encoding Neutralization-Related Epitopes

Abstract: Nucleotide sequences of a part of the envelope glycoprotein B (gB) gene of human cytomegalovirus (CMV), encoding epitopes recognized by virus-neutralizing monoclonal antibodies, were determined for 12 distinct clinical strains of CMV after amplification of suitable templates using the polymerase chain reaction. Sequence analysis of this region (codons 384-717) revealed that the clinical strains and previously sequenced laboratory strains Towne and AD169 belong to one of four variant groups, each with a characteristic nucleotide and peptide sequence. Peptide homology was greater than 99% for strains within a group, and varied from 91% to 98% for strains in different groups. Variation was most frequent between codons 448 and 480. The gB group of a CMV strain could be determined by restriction analysis of a small target sequence amplified from viral genomic DNA, and an additional 28 clinical strains were grouped in this manner. The existence of a limited number of variants of gB among clinical strains facilitates analysis of biologic function and cross-reactivity of immune responses.

Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae

Abstract: DNA fragments (generated by BamHI treatment) with no homology to the yeast genome were transformed into Saccharomyces cerevisiae. When the fragments were transformed in the presence of the BamHI enzyme, they integrated into genomic BamHI sites. When the fragments were transformed in the absence of the enzyme, they integrated into genomic G-A-T-C sites. Since the G-A-T-C sequence is present at the ends of BamHI fragments, this results indicates that four base pairs of homology are sufficient for some types of mitotic recombination.

DNA of Saccharomyces cerevisiae.

Abstract: Publisher Summary This chapter describes the DNA of Saccharomyces cerevisiae strains used in genetic studies. Discussion of DNA isolation procedures is followed by a description of useful information derived from analysis of restriction spectra. Interpretation of the band patterns can be quite useful. First, the extent of cleavage (partial or complete) can be deduced for many restriction endonucleases. Second, the average copy number of newly introduced plasmids can be estimated. Third, restriction site polymorphisms in repeated DNA can be detected. The sequence organization and the location of restriction sites for the four classes of repeated DNA (rDNA, 2-μm plasmid, Ty elements, telomeric Y' sequences) which are the origin of most bands seen in restriction spectra are discussed in the chapter. The ribosomal DNA consists of a cluster of usually 100 to 120 tandem copies of a 9.08-kb repeat unit. Cell clones with higher and lower copy number are observed.

Direct selection: a method for the isolation of cDNAs encoded by large genomic regions.

Abstract: We have developed a strategy for the rapid enrichment and identification of cDNAs encoded by large genomic regions. The basis of this "direct selection" scheme is the hybridization of an entire library of cDNAs to an immobilized genomic clone. Nonspecific hybrids are eliminated and selected cDNAs are eluted. These molecules are then amplified and are either cloned or subjected to further selection/amplification cycles. This scheme was tested using a 550-kilobase yeast artificial chromosome clone that contains the EPO gene. Using this clone and a fetal kidney cDNA library, we have achieved a 1000-fold enrichment of EPO cDNAs in one cycle of enrichment. More significantly, we have further investigated one of the "anonymous" cDNAs that was selectively enriched. We confirmed that this cDNA was encoded by the yeast artificial chromosome. Its frequency in the starting library was 1 in 1 x 10(5) cDNAs and after selection comprised 2% of the selected library. DNA sequence analysis of this cDNA and of the yeast artificial chromosome clone revealed that this gene encodes the beta 2 subunit of the human guanine nucleotide-binding regulatory proteins. Restriction mapping and hybridization data position this gene (GNB2) to within 30-70 kilobases of the EPO gene. The selective isolation and mapping of GNB2 confirms the feasibility of this direct selection strategy and suggests that it will be useful for the rapid isolation of cDNAs, including disease-related genes, across extensive portions of the human genome.

Polymerase chain reaction for detection of Mycoplasma genitalium in clinical samples.

Abstract: We have used the polymerase chain reaction to detect Mycoplasma genitalium in artificially seeded human throat swab samples as well as in clinical material. On the basis of the published nucleotide sequence of the M. genitalium 140-kDa adhesin gene, primers were chosen to produce an amplified fragment of 281 bp. Five different previously isolated strains, including the type strain of M. genitalium, could all be detected by the polymerase chain reaction, and DNAs from other mycoplasmal and bacterial species yielded negative results. The detection limits were estimated to be approximately 50 organisms by inspection of ethidium bromide-stained agarose gels and 4 organisms when a biotinylated oligonucleotide probe was used in filter hybridization. The amplified DNA fragments were subjected to restriction enzyme analysis. DNAs from the five different isolates all possessed EcoRI, SspI, AluI, Sau3AI, and DdeI restriction sites, as predicted from the published sequence. A total of 150 urogenital swabs collected from 100 patients for culturing of Chlamydia trachomatis were tested for the presence of M. genitalium DNA. Ten samples from eight patients were found positive. The amplified DNA fragments from all of our clinical samples possessed the AluI, Sau3AI, and DdeI restriction sites, but three samples from two patients did not contain the SspI site and none of the samples contained the EcoRI site.

Several groups among human herpesvirus 6 strains can be distinguished by Southern blotting and polymerase chain reaction.

Abstract: Eight human herpesvirus 6 (HHV-6) strains were studied by Southern blot and polymerase chain reaction. DNA from infected cells was digested by a panel of restriction enzymes and hybridized with cloned BamHI fragments corresponding to about 30% of the HHV-6 strain SIE genome. In parallel, this DNA was amplified by polymerase chain reaction using pairs of primers derived from the strain SIE nucleotide sequence. Subsequently, amplification products were analyzed by hybridization, digestion with restriction endonucleases, and partial nucleotide sequencing. Overall results indicated that all strains were closely related to one another. However, concordant differences in restriction patterns allowed at least two groups to be distinguished, typified by strains SIE and HST, respectively. Differences between the two groups were found to reflect a limited number of punctual changes in nucleotide sequences. These results strengthen the idea of a unique HHV-6 species with genetic polymorphism. In addition, this study provides useful markers for the diagnosis and molecular epidemiology of HHV-6 infections. Images

Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate.

Abstract: The pathogenic yeast, Candida albicans, is insensitive to the anti-mitotic drug, benomyl, and to the dihydrofolate reductase inhibitor, methotrexate. Genes responsible for the intrinsic drug resistance were sought by transforming Saccharomyces cerevisiae, a yeast sensitive to both drugs, with genomic C. albicans libraries and screening on benomyl or methotrexate. Restriction analysis of plasmids isolated from benomyl- and methotrexate-resistant colonies indicated that both phenotypes were encoded by the same DNA fragment. Sequence analysis showed that the fragments were nearly identical and contained a long open reading frame of 1694 bp (ORF1) and a small ORF of 446 bp (ORF2) within ORF1 on the opposite strand. By site-directed mutagenesis, it was shown that ORF1 encoded both phenotypes. The protein had no sequence similarity to any known proteins, including β-tubulin, dihydrofolate reductase, and the P-glycoprotein of the multi-drug resistance family. The resistance gene was detected in several C. albicans strains and in C. stellatoidea by DNA hybridization and by the polymerase chain reaction.

The peptide antibiotic microcin B17 induces double-strand cleavage of DNA mediated by E. coli DNA gyrase.

Abstract: Microcin B17 (MccB17) is a bactericidal peptide antibiotic which inhibits DNA replication. Two Escherichia coli MccB17 resistant mutants were isolated and the mutations were shown to map to 83 min of the genetic map. Cloning of the mutations and Tn5 insertional analysis demonstrated that they were located inside gyrB. The approximate location of the mutations within gyrB was determined by constructing hybrid genes, as a previous step to sequencing. Both mutations were shown to consist of a single AT----GC transition at position 2251 of the gene, which produces a Trp751----Arg substitution in the amino acid sequence of the GyrB polypeptide. The inhibitory effect of MccB17 on replicative cell-free extracts was assayed. In this in vitro system, interaction of MccB17 with a component of the extracts induced double-strand cleavage of plasmid DNA. In vivo treatment with MccB17 also induced a well-defined cleavage pattern on chromosomal DNA. These effects were not observed with a MccB17-resistant, gyrB mutant. Altogether, our results indicate that MccB17 blocks DNA gyrase by trapping an enzyme-DNA cleavable complex. Thus, the mode of action of this peptide antibiotic resembles that of quinolones and a variety of antitumour drugs currently used in cancer chemotherapy. MccB17 is the first peptide shown to inhibit a type II DNA topoisomerase.

Interstrand cross-links are preferentially formed at the d(GC) sites in the reaction between cis-diamminedichloroplatinum (II) and DNA.

Abstract: A DNA restriction fragment with convergent SP6 and T7 promoters has undergone reaction with cis-diamminedichloroplatinum(II) (cis-DDP) and was then used as a template for RNA synthesis in vitro. The T7 and SP6 RNA polymerases generate fragments of defined sizes. Analysis of the RNA fragments shows that the polymerases are mainly blocked at the level of the d(GG) and d(AG) sites and to a lesser extent at the level of the d(GC) sites. The adducts at the d(GC) sites are more resistant to cyanide ion attack than those at the major sites and are identified as interstrand cross-links. The formation of an interstrand cross-link between the N-7 atoms of two guanine residues at the d(GC) sites was further confirmed by chemical modifications.

Molecular analysis of the Escherichia coli hns gene encoding a DNA-binding protein, which preferentially recognizes curved DNA sequences.

Abstract: We previously demonstrated that the E. coli protein, H-NS (or H1a), encoded by the gene hns (or osmZ or bglY) preferentially recognizes curved DNA sequences in vitro. In order to gain further insight into the complex function of H-NS and the significance of DNA curvature, we constructed a structurally defined hns deletion mutant on the E. coli chromosome. The hns deletion mutant thus obtained showed a variety of phenotypes previously for other lesions in hns. It was further demonstrated that, in this hns deletion background, numerous E. coli cellular proteins were either strongly expressed or remarkably repressed, as compared to their expression levels in wild-type cells.

DNA fingerprinting of Enterococcus faecium by pulsed-field gel electrophoresis may be a useful epidemiologic tool.

Abstract: Pulsed-field gel electrophoresis was used to compare 34 isolates of Enterococcus faecium from six different geographic locations. This procedure generated an average of 13 discernible fragment bands per isolate (range, 10 to 19 fragment bands) of 34 to 485 kb. The resulting restriction endonuclease digestion patterns were quite heterogeneous and were able to differentiate 27 of 34 isolates from each other, as defined by one or more mismatched fragment bands. Five patterns were shared by two or more isolates, and each set of isolates with matching patterns (shared pattern) originated in the same medical center, suggesting a common epidemiologic background, including highly penicillin resistant isolates in Richmond and Philadelphia. We conclude that pulsed-field gel electrophoresis of DNA digested with low-frequency-cleavage restriction enzymes offers a relatively simple method of comparing E. faecium for the purpose of epidemiologic study.

Genetic transformation in Streptococcus pneumoniae: nucleotide sequence analysis shows comA, a gene required for competence induction, to be a member of the bacterial ATP-dependent transport protein family.

Abstract: The complete nucleotide sequence of comA, a gene required for induction of competence for genetic transformation in Streptococcus pneumoniae, was determined by using plasmid DNA templates and synthetic oligonucleotide primers. The sequence contained a single large open reading frame, ORF1, of 2,151 bp. ORF1 was included within the comAB locus previously mapped genetically and accounted for 50% of its extent. The predicted molecular weight of the largest polypeptide encoded within ORF1, 80,290, coincided with that measured previously (77,000) for the product of in vitro transcription-translation of the cloned comA locus. A Shine-Dalgarno sequence (AAAGGAG, delta G = -14 kcal) lay immediately upstream of ORF1. A sequence (TTtAat-17 bp-TAaAAT) similar to the Escherichia coli sigma 70 promoter consensus was located 410 bp upstream of ORF1. The deduced protein sequence of ComA showed a very strong similarity to the E. coli hemolysin secretion protein, HlyB, and strong similarities to other members of the family of ATP-dependent transport proteins, including the mammalian multidrug resistance P-glycoprotein. These similarities suggest that ComA functions in the transport of some molecule, possibly pneumococcal competence factor itself.

Mapping of replication initiation sites in mammalian genomes by two-dimensional gel analysis: stabilization and enrichment of replication intermediates by isolation on the nuclear matrix.

Abstract: Two complementary two-dimensional gel electrophoretic techniques have recently been developed that allow initiation sites to be mapped with relative precision in eukaryotic genomes at least as complex as those of yeast and Drosophila melanogaster. We reported the first application of these mapping methods to a mammalian genome in a study on the amplified dihydrofolate reductase (DHFR) domain of the methotrexate-resistant CHO cell line CHOC 400 (J.P. Vaughn, P.A. Dijkwel, and J.L. Hamlin, Cell 61:1075-1087, 1990). Our results suggested that in this 240-kb domain, initiation of nascent DNA strands occurs at many sites within a 30- to 35-kb zone mapping immediately downstream from the DHFR gene. In the course of these studies, it was necessary to develop methods to stabilize replication intermediates against branch migration and shear. This report describes these stabilization methods in detail and presents a new enrichment protocol that extends the neutral/neutral two-dimensional gel mapping method to single-copy loci in mammalian cells. Preliminary analysis of replication intermediates purified from CHO cells by this method suggests that DNA synthesis may initiate at many sites within a broad zone in the single-copy DHFR locus as well.

A family of regulatory genes associated with type II restriction-modification systems.

Abstract: Restriction-modification systems must be regulated to avoid autorestriction and death of the host cell. An open reading frame (ORF) in the PvuII restriction-modification system appears to code for a regulatory protein from a previously unrecognized family. First, interruptions of this ORF result in a nonrestricting phenotype. Second, this ORF can restore restriction competence to such interrupted mutants in trans. Third, the predicted amino acid sequence of this ORF resembles those of known DNA-binding proteins and includes a probable helix-turn-helix motif. A survey of unattributed ORFs in 15 other type II restriction-modification systems revealed three that closely resemble the PvuII ORF. All four members of this putative regulatory gene family have a common position relative to the endonuclease genes, suggesting a common regulatory mechanism.

Physical map of the Saccharomyces cerevisiae genome at 110-kilobase resolution.

Abstract: A physical map of the Saccharomyces cerevisiae genome is presented. It was derived by mapping the sites for two restriction endonucleases, SfiI and NotI, each of which recognizes an 8-bp sequence. DNA-DNA hybridization probes for genetically mapped genes and probes that span particular SfiI and NotI sites were used to construct a map that contains 131 physical landmarks--32 chromosome ends, 61 SfiI sites and 38 NotI sites. These landmarks are distributed throughout the non-rDNA component of the yeast genome, which comprises 12.5 Mbp of DNA. The physical map suggests that those genes that can be detected and mapped by standard genetic methods are distributed rather uniformly over the full physical extent of the yeast genome. The map has immediate applications to the mapping of genes for which single-copy DNA-DNA hybridization probes are available.

Novel structure of the recA locus of Mycobacterium tuberculosis implies processing of the gene product.

Abstract: A fragment of Mycobacterium tuberculosis DNA containing recA-like sequences was identified by hybridization with the Escherichia coli recA gene and cloned. Although no expression was detected from its own promoter in E. coli, expression from a vector promoter partially complemented E. coli recA mutants for recombination, DNA repair, and mutagenesis, but not for induction of phage lambda. This clone produced a protein which cross-reacts with antisera raised against the E. coli RecA protein and was approximately the same size. However, the nucleotide sequence of the cloned fragment revealed the presence of an open reading frame for a protein about twice the size of other RecA proteins and the cloned product detected by Western blotting (immunoblotting). The predicted M. tuberculosis RecA protein sequence was homologous with RecA sequences from other bacteria, but this homology was not dispersed; rather it was localized to the first 254 and the last 96 amino acids, with the intervening 440 amino acids being unrelated. Furthermore, the junctions of homology were in register with the uninterrupted sequence of the E. coli RecA protein. Identical restriction fragments were found in the genomic DNAs of M. tuberculosis H37Rv and H37Ra and of M. bovis BCG. It is concluded that the ancestral recA gene of these species diversified via an insertional mutation of at least 1,320 bp of DNA. Possible processing mechanisms for synthesizing a normal-size RecA protein from this elongated sequence are discussed.