Identical Cleavage Patterns Research Articles

Mycobacterium aviuminfection in a farmed deer herd

Tuberculous lesions were identified over a 2-year period in 36 clinically normal red deer from a single herd. The lesions were only present in the retropharyngeal lymph nodes and lymph nodes draining the intestinal tract, indicating infection by the oral route. Mycobacterium avium was isolated from 27 of 29 lesions examined by bacterial culture. Grossly and histologically, the lesions were indistinguishable from those caused by Mycobacterium bovis. DNA restriction endonuclease analysis revealed that all the 26 M. avium isolates available for examination had identical cleavage patterns. These patterns were identical to a New Zealand M. avium serotype 2 isolate from a pig and were very similar to a reference strain of M. avium serotype 2. The DNA examinations indicated that the deer were infected from a common source that was not identified.

Role of the D arm and the anticodon arm in tRNA recognition by eubacterial and eukaryotic RNase P enzymes.

Truncated precursor tRNAs lacking the D arm or anticodon arm were studied in vitro as substrates for RNase P enzymes from Escherichia coli, Thermus thermophilus (eubacteria), and HeLa. Deletion of the D arm still allowed 5'-processing by E. coli RNase P, but strongly impaired maturation by T. thermophilus and HeLa extracts. In contrast, deletion of the anticodon arm had no influence on processing by RNase P activities from all three organisms. Inhibition kinetics and gel retardation studies showed that deletion of the D arm leads to low-affinity binding to E. coli RNase P RNA (M1 RNA). However, the E. coli enzyme appears to form sufficiently strong contacts in the region of the T arm, acceptor stem, and CCA terminus to still allow productive enzyme-substrate interaction even in the absence of the structural contribution provided by the D arm. Pb(2+)-induced hydrolysis of a tRNAGly from T. thermophilus gave identical cleavage patterns in the D arm and anticodon loop in the absence and presence of E. coli M1 RNA, whereas lead hydrolysis was strongly reduced at the CUCCAA 3'-terminus due to the presence of the enzyme.

The calcium pump of the liver nuclear membrane is identical to that of endoplasmic reticulum.

The envelope membrane of rat liver nuclei contains a P-type Ca(2+)-transporting pump, revealed by the presence of a Ca(2+)-stimulated phosphoenzyme. The level of the nuclear phosphoenzyme in autoradiographed polyacrylamide gels was decreased by lanthanum, as typically observed in the endoplasmic reticulum Ca2+ pump. It was also decreased by thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone, two accepted inhibitors of the endoplasmic reticulum Ca(2+)-ATPase. Comparative proteolysis of the phosphorylated enzyme of liver microsomes (endoplasmic reticulum) and nuclear membranes revealed an identical cleavage pattern. In addition, antibodies raised against the endoplasmic reticulum Ca2+ pump cross-reacted with the pump in the nuclear membranes. The findings show that nuclear membranes contain a Ca(2+)-transporting pump closely related to that of the endoplasmic reticulum, if not identical to it. The pump is likely to be involved in the control of nuclear free calcium.

A role for the metal binding domain in determining the DNA sequence selectivity of Fe-bleomycin.

Previous studies of Fe-bleomycin-mediated DNA cleavage have established that the bithiazole moiety + C-terminal substituent of bleomycin are required for DNA binding, while the metal binding domain is responsible for O2 activation. Although recent studies have indicated that the metal binding domain also participates in DNA unwinding, and in determining the sequence and strand selectivity of DNA cleavage, no study has defined the structural domain that bears primary responsibility for the observed pattern of bleomycin-mediated DNA degradation. Presently, by the use of four synthetic analogs of bleomycin demethyl A2 having the functional domains connected by rigid spacers of varying lengths, the source of DNA cleavage specificity has been determined. When the four analogs cleaved 242- and 127-base pair 5'-32P-end-labeled DNA restriction fragments containing isolated Fe-bleomycin cleavage sites, all four produced cleavage at the same preferred sites. Because the (oligo)glycine spacers altered the distance between the domains by as much as 14 A, the identical cleavage patterns argue that the primary determinant of sequence specificity for these analogs is the metal binding domain.

Probing the environment of lanthanide binding sites in yeast tRNA(Phe) by specific metal-ion-promoted cleavages.

Specific yeast tRNA(Phe) hydrolysis brought about by europium ions has been studied in detail using the 32P-end-labeled tRNA and polyacrylamide gel electrophoresis. The dependence of the induced cleavages on pH, temperature and concentration of the europium ions has been determined. Europium hydrolyzes yeast tRNA(Phe) in the D-loop at phosphates 16 and 18, and the anticodon loop of phosphates 34 and 36. The two D-loop cuts are thought to take place from two distinct europium binding sites, while the two anticodon loop cleavages from a single site. Eight other members of the lanthanide series and ytrium give basically the same pattern of cleavages as europium. The specific cleavages taking place in the anticodon loop occur in an intramolecular mode from the lanthanide binding site that has not been found in yeast tRNA(Phe) crystal structure. It appears from the comparison of the europium-promoted cuts with those generated by magnesium and lead that the former two ions give more similar but not identical cleavage patterns. The usefulness of the specific cleavages induced by lanthanides for probing their own and magnesium binding sites in tRNA is discussed.

The Two Xenopus laevis SRC Genes Are Co-expressed and Each Produces Functional pp60src

Abstract The haploid genome of Xenopus laevis contains two src genes, and transcripts from both genes are found in the maternal RNA pool of the oocyte (Steele, R. E. (1985) Nucleic Acids Res. 13, 1747-1761). We have now isolated cDNA clones which contain complete coding sequences from both src mRNAs. In vitro translation of RNAs transcribed in vitro from these clones produces in each case a protein with an apparent molecular mass of 57 kDa. The in vitro-synthesized proteins show identical protease cleavage patterns. Sequence analysis of the coding regions of the two cDNAs revealed that they both produce 532-amino acid polypeptides which differ from each other at only eight sites. Analysis of silent site changes between the two coding sequences suggests that the two genes began diverging about 25 million years ago. Hybridization with probes specific for each of the two src RNAs indicates that the two genes are co-expressed in embryos and in at least some adult tissues as well as during oogenesis. Finally, expression of each of the cDNA clones in yeast causes the appearance of proteins which are recognized by an antibody which binds to phosphotryosine.

Cation-mediated release and proteolytic cleavage of basic protein of isolated human myelin at acid pH

Myelin from human brain was incubated at pH 4.4 with metal salts, including KCl, NaCl, CaCl2, and MgSO4, to elicit cation-dependent autoproteolysis of myelin proteins. Incubation of myelin resulted in soluble proteolytic breakdown products of Mr smaller than those of the three original myelin basic proteins (MBPs). Comparable polypeptides were essentially absent from residual myelin. Proteolysis was strongly stimulated by increasing millimolar concentrations of K+, Na+, and Mg2+ and only moderately by Ca2+. Breakdown products were traced to MBP by immunostaining. Their origin from MBP was also indicated by identical electrophoretic cleavage patterns from endogenous myelin protein and exogenous MBP. All four metal salts, in addition to activating endogenous proteolysis, also caused a biphasic extraction of MBP. Electrophoresis of myelin revealed a quick initial and a slow further loss of protein, eventually leading to the removal of up to 78% of original MBP. The results are consistent with a concurrent extraction of MBP and activation of latent-bound acid protease activity by metal cations. It is therefore suggested that, in particular disease states, unfavorable changes in electrolytes and pH of white matter may cause a selective loss and proteolytic cleavage of MBP.

Restriction Endonuclease Patterns of Some European and American Isolates of Avian Infectious Laryngotracheitis Virus

Eleven isolates of infectious laryngotracheitis virus (nine European and two American) were compared by restriction endonuclease analysis of their DNA after radiolabeling with 32P. Digestion with KpnI gave identical cleavage patterns for all the European isolates, but the two American viruses (one field and one vaccine) showed some differences from them and from each other. In the case of the American vaccine strain, however, these differences were only minor. After BamHI digestion, only the American field isolate appeared to be different, whereas with HindIII, all 11 isolates were identical.

Maternal stores of α subtype histone mRNAs are not required for normal early development of sea urchin embryos.

The ability to specifically delete the store of maternal α-subtype histone mRNAs stored in the egg pronucleus has allowed us to examine the role of this major fraction of the maternal mRNA in the early development of the sea urchinStrongylocentrotus purpuratus. The egg nucleus was removed by centrifugation, and the resulting enucleate half eggs were fertilized. These haploid andromerogones lacked any stored α-subtype histone mRNAs. However, when grown in parallel with control embryos, they showed identical cleavage cycles, cell numbers, and patterns of cell differentiation. Measurements of the amount of α-histone mRNA in these andromerogones showed that there was no premature synthesis of α-histone mRNAs to compensate for the deleted maternal pool. Instead embryonic synthesis was normal in timing of initiation and duration. the ability of these embryos to develop into highly differentiated larvae without their maternal α-subtype histone mRNA pool suggests that this pool is not a critical component of early development per se. This suggestion is strengthened by the observation that the primitive sea urchinEucidaris tribuloides naturally lacks this maternal histone mRNA store. Evolutionary implications are discussed.

Biosynthesis of two developmentally distinct acid phosphatase isozymes in Dictyostelium discoideum.

The presence of a common antigenic determinant on the Dictyostelium discoideum acid phosphatase isozyme 1 (ap 1), and the absence of this determinant on the isozyme ap2 enables separation of the two isozymes. This separation is accomplished by removal of ap1 from samples with a common antigen monoclonal antibody followed by immunoprecipitation of ap2 with an acid phosphatase monoclonal antibody. Application of this separation scheme on cells pulse-labeled early (2 h) and late (18 h) in the developmental cycle reveal that ap1 protein synthesis occurs only early in development and that the protein remains stable throughout development, whereas ap2 protein synthesis occurs only late in development. Furthermore, pulse-chase experiments during both early and late development reveal that both isozymes of acid phosphatase are initially synthesized as precursor molecules (Mr = 60,000) which are then processed to mature forms (Mr = 58,000). The processing event(s) for acid phosphatase begin in less than 5 min compared to 25-30 min for Dictyostelium alpha-mannosidase and 10-15 min for Dictyostelium beta-glucosidase. Endoglycosidase H and Endoglycosidase F treatment of both isozymes reveals identical cleavage patterns for ap1 and ap2, indicating that the amount of carbohydrate on both molecules is equivalent. Preliminary studies to identify modification differences reveal that fucose is not present on either isozyme; however, sulfate is present on the ap1 isozyme and absent on the ap2 isozyme. These results suggest that differences in the modification of newly synthesized acid phosphatase at different times during the Dictyostelium life cycle result in the appearance of two distinct acid phosphatase isozymes.

Restriction endonuclease analysis of cytomegalovirus DNA from strains isolated in day care centers.

Cytomegalovirus (CMV) strains isolated in a previous study from children in group day care have been analyzed by restriction endonuclease cleavage of DNA. CMV was isolated from 16 of 60 children (27%) in several centers in Stockholm. In one center (Center A) 7 of 20 children excreted CMV and all were in the same group. In another center (Center B) 5 of 16 children were CMV-positive. In Center A three children excreted CMV strains with identical DNA cleavage patterns. The other four strains had different unique patterns as had those isolated in Center B. This finding provides evidence that CMV can be transmitted between children in group day care.

Functional characterization of thermolabile DNA-binding proteins that affect adenovirus DNA replication.

The human adenovirus type 2 (Ad2) mutant Ad2ts111 has previously been shown to contain two mutations which result in a complex phenotype. Ad2ts111 contains a single base change in the early region 1B (E1B) 19,000-molecular-weight (19K) coding region which yields a cyt deg phenotype and another defect which maps to the E2A 72K DNA-binding protein (DBP) coding region that causes a temperature-sensitive DNA replication phenotype. Here we report that the defect in the Ad2ts111 DBP is due to a single G----T transversion that results in a substitution of valine for glycine at amino acid 280. A temperature-independent revertant, Ad2ts111R10, was isolated, which reverts back to glycine at amino acid 280 yet retains the cyt and deg phenotypes caused by the 19K mutation. We physically separated the two mutations of Ad2ts111 by constructing a recombinant virus, Ad2ts111A, which contained a wild-type Ad2 E1B 19K gene and the gly----val mutation in the 72K gene. Ad2ts111A was cyt+ deg+, yet it was still defective for DNA replication at the nonpermissive temperature. The Ad2ts111 DBP mutation is located only two amino acids away from the site of the mutation in Ad2+ND1ts23, a previously sequenced DBP mutant. Biochemical studies of purified Ad2+ND1ts23 DBP showed that this protein was defective for elongation but not initiation of replication in a cell-free replication system consisting of purified Ad polymerase, terminal protein precursor, and nuclear factor I. Ad2+ND1ts23 DBP bound less tightly to single-strand DNA than did Ad2 DBP, as shown by salt gradient elution of purified DBPs from denatured DNA cellulose columns. This decreased binding to DNA was probably due to local conformational changes in the protein at a site that is critical for DNA binding rather than to global changes in protein structure, since both the Ad2+ND1ts23 and Ad2 DBPs showed identical cleavage patterns by the protease thermolysin at various temperatures.

Viral DNA sequences detected in a hamster liposarcoma induced by bovine papillomavirus type 4.

Following intradermal inoculation of bovine papillomavirus type 4 (BPV-4) into a Syrian hamster, a liposarcoma developed at the inoculation site 20 months later. The DNA of this tumour contained multiple copies of the BPV-4 genome which existed in a free unintegrated state. Unintegrated viral DNA and viral DNA isolated from virus particles from bovine alimentary tract papillomas revealed identical cleavage patterns with CpG methylation-resistant and -sensitive restriction enzymes: apparently there was no gross methylation of CpG sites in either case. The entire BPV-4 genome appeared to be represented in the tumour DNA.

Changes in site specificity of single-strand-specific endonucleases on supercoiled PM2 DNA with temperature and ionic environment.

Mung bean nuclease sites in supercoiled PM2 DNA at neutral pH were located by linearizing the singly-nicked circular DNA product with venom phosphodiesterase followed by restriction endonuclease mapping. The locations of the sites varied with small changes in temperature and in concentration of NaC1 or magnesium ion. Different environmental changes which affect duplex stability in the same direction showed similar effects on the number of sites and in some cases resulted in identical cleavage patterns. Venom phosphodiesterase and P1 nuclease showed cleavage patterns similar to mung bean nuclease under the same environmental conditions and showed similar variations in cleavage patterns when environmental conditions were changed. Relaxed, closed-circular DNA was slowly cleaved at numerous sites whose locations did not vary with environment. Changes in site specificity are likely the result of environmental effects on the conformation of supercoiled DNA as opposed to effects on the single-strand-specific endonucleases themselves.

DNA cleavage patterns as indicators of genotypic heterogeneity among strains of Acholeplasma and Mycoplasma species.

Electrophoretic patterns of digestion products of Acholeplasma and Mycoplasma DNA by restriction endonucleases were compared. The patterns of Acholeplasma axanthum strains isolated from a variety of hosts and habitats differed markedly from each other, indicating considerable genotypic heterogeneity among strains included in this species. Heterogeneity was less marked among the Acholeplasma oculi strains tested, and was minimal among strains of the avian pathogen Mycoplasma gallisepticum. Strains of Mycoplasma genitalium isolated from the urethra of patients with non-gonococcal urethritis and from the urethra of an experimentally infected chimpanzee yielded identical cleavage patterns, indicating a high degree of genetic homogeneity of these strains. The data support the notion that mycoplasma species of strict host and tissue specificity exhibit marked genetic homogeneity. The advantages and deficiencies of the use of DNA cleavage patterns for classification purposes are discussed.

Relationship between laboratory mice and the subspecies Mus musculus domesticus based on restriction endonuclease cleavage patterns of mitochondrial DNA.

The mitochondrial DNAs of house mouse, Mus musculus in twenty-five different laboratory strains showed identical cleavage patterns on digestion with nine different restriction endonucleases. The cleavage patterns were identical to those of Canadian wild mice, M. m. domesticus but clearly different from the patterns of three Asian subspecies, M. m. bactrianus, M. m. castaneus and M. m. molossinus. Based on comparison of the cleavage maps of the four subspecies, the time of divergence between M. m. domesticus and three Asian subspecies was estimated to be 1-2.5×106 years. These results suggest that the genetic background of laboratory mice is mainly derived from M. m. domesticus.

Occurrence of a class II restriction endonuclease in Staphylococcus aureus.

The occurrence of class II restriction endonucleases (enzymes that both recognize and cleave a specific nucleotide sequence in deoxyribonucleic acid [DNA] in Staphylococcus aureus has been investigated by analysis of crude extracts obtained from different propagating strains of the International Phage Typing System. Of the four main groups of strains in the International System, only extracts of group II strains were found to contain class II restriction endonucleases. The identical cleavage patterns obtained by incubation of different DNAs with cell extracts of group II strains suggest that these enzymes all recognize and cleave the same nucleotide sequence. This recognition site has been determined to be 5'-G-A-T-C-3'-3'-C-T-A-G-5' for the prototype of these enzymes, Sau3AI (J. S. Sussenbach et al., Nucleic Acids Res. 3:3192-3202, 1976). Evidence is presented that the classification of group II strains is based on restriction modification and is correlated with the presence of a class II restriction enoduclease that recognizes and cleaves the above sequence.

Human papilloma virus DNA: physical mapping and genetic heterogeneity.

The molecular weight of three preparations of human papilloma virus DNA derived from different plantar warts was determined by agarose gel electrophoresis or electron microscopic contour length measurement. It was found to amount to approximately 4.9 X 10(6). Analysis of this DNA after sequential digestion by four different restriction endonucleases (EcoRI, Bam, Hind II, and Hind III) permitted physical mapping of the cleavage sites. Two of the three DNA preparations revealed an identical cleavage pattern, whereas the third one contained two additional cleavage sites.