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S M Halling

Publications and source records attributed to S M Halling.

32 records · Page 2Linked to original sources
PubMed1990

Polymorphism in Brucella spp. due to highly repeated DNA.

The species of Brucella are very closely related, but Brucella ovis does not express detectable amounts of a protein, designated BCSP31, that is common to the other species. We studied the lack of expression of BCSP31 by Southern analysis. DNAs from the B. ovis culture collection strains and field isolates were probed with a 1.3-kb HindIII fragment encoding BCSP31 of Brucella abortus. The probe hybridized to a 1.6-kb HindIII fragment of all B. ovis strains tested, showing that the gene is present in B. ovis but occurs on a larger restriction fragment. DNA linkage studies and restriction mapping of the cloned polymorphic region of B. ovis showed that the polymorphism was due to a DNA insertion of approximately 0.9 kb at a site downstream of the BCSP31-coding region. When the 1.6-kb polymorphic B. ovis fragment was used to probe a HindIII Southern blot of cellular DNA of strains of B. ovis and of B. abortus, at least 24 fragments of B. ovis and 6 fragments of B. abortus hybridized to the inserted DNA. Specimens of B. ovis collected over a 30-year period on two continents had similar hybridization patterns. The large difference between B. ovis and B. abortus in the number of copies of the repeated DNA is interesting in the context of the closeness of the Brucella species.

Blotting, Southern↗
PubMed1988

The cloning, expression, and nucleotide sequence of a gene coding for an immunogenic Brucella abortus protein.

Brucella abortus is the causative agent for brucellosis in cattle and man. Development of a single diagnostic test for the differentiation of vaccinated from infected animals and the development of a nonviable 'subunit' vaccine are top priorities of the brucellosis research program in the United States. Preliminary evidence previously showed that a purified 31-kDa protein (thought to be localized at or near the bacterial cell surface) protects against experimental brucellosis in rodents. The gene for this 31-kDa protein has now been cloned in Escherichia coli. The protein is expressed well, apparently from its native promoter, when placed in several different E. coli plasmids. The nucleotide sequence of the flanking and encoding sequences has been determined, and comparison with the N-terminal amino acid (aa) sequence of the mature protein indicates the presence of a putative 28-aa signal sequence. The availability of the 31-kDa protein free of Brucella contaminants now allows rigorous study of the immunological properties of this protein.

Amino Acid Sequence↗
PubMed1984

Expression of canine parvovirus-beta-galactosidase fusion proteins in Escherichia coli.

Cloned DNA fragments encoding portions of canine parvovirus (CPV) structural proteins were inserted into plasmid expression vectors. These plasmids expressed CPV-beta-galactosidase fusion proteins under the transcriptional control of the Escherichia coli lac promoter-operator. The fusion proteins were purified and used to immunize rabbits. Rabbit antibodies raised against these fusion proteins were shown to immunoprecipitate authentic CPV structural proteins from infected cell extracts. This demonstrated that the CPV-beta-galactosidase fusion proteins expressed in bacteria elicit antibodies which can recognize determinants of authentic CPV proteins. However, none of the antibodies neutralizes CPV virus particles.

Animals↗
PubMed1982

A symmetrical six-base-pair target site sequence determines Tn10 insertion specificity.

Transposon Tn10 inserts at many sites in the bacterial chromosome, but preferentially inserts at particular hotspots. We believe we have identified the target DNA signal responsible for this specificity. We have determined the DNA sequences of 11 Tn10 insertion sites and identified a particular 6 base pair (bp) symmetrical consensus sequence (GCTNAGC) common to those sites. The sequences at some sites differ from the consensus sequence but only in limited and well defined ways. The sequences at some sites differ from the consensus sequence than do sequences at other sites, and the consensus sequence and closely related sequences are generally absent from potential target regions where Tn10 is known not to insert. Other aspects of the target DNA can significantly influence the efficiency with which a particular target site sequence is used. The 6 bp consensus sequence is symmetrically located within the 9 bp target DNA sequence that is cleaved and duplicated during Tn10 insertion. This juxtaposition of recognition and cleavage sites plus the symmetry of the perfect consensus sequence suggest that the target DNA may be both recognized and cleaved by the symmetrically disposed subunits of a single protein, as suggested for type II restriction endonucleases. There is plausible homology between the consensus sequence and the very ends of Tn10, compatible with recognition of transposon ends and target DNA by the same protein. The sequences of actual insertion sites deviate from the perfect consensus sequence in a way which suggests that the 6 bp specificity determinant may be recognized through protein-DNA contacts along the major groove of the DNA double helix.

Base Sequence↗
PubMed1982

DNA sequence organization of IS10-right of Tn10 and comparison with IS10-left.

Tn10 is 9,300 base pairs long and has inverted repeats of an insertion sequence (IS)-like sequence (IS10) at its ends. IS10-right provides all of the Tn10-encoded functions used for normal Tn10 transposition. IS10-left can also provide these functions but at a much reduced level. We report here the complete nucleotide sequence of IS10-right and a partial sequence of IS10-left. From our analysis of this information, we draw the following conclusions. (i) IS10-right is 1,329 base pairs long. Like most IS elements, it has short (23-base pair) nearly perfect inverted repeats at its termini. We can divide these 23-base pair segments into at least two functionally distinct parts. IS10-right also shares with other elements the presence of a single long coding region that extends the entire length of the element. Genetic evidence suggests that this coding region specifies an essential IS10 transposition function. A second, overlapping, coding region may or may not be important. (ii) The "outside" end of IS10-right contains three suggestively positioned internal symmetries. Two of these (A1 and A2) are nearly identical in sequence. Symmetry A1 overlaps the terminal inverted repeat; symmetry A2 overlaps the promoter shown elsewhere to be responsible for expression of IS10 functions and lies very near a second characterized promoter that directs transcription outward across the end of IS10. Symmetries A1 and A2 may play a role in modulation of Tn10 activity and are likely to function at least in part as protein recognition sites. We propose that the third symmetry (B) acts to prevent fortuitous expression of IS10 functions from external promoters. The transcripts from such promoters can assume a stable secondary structure in which the AUG start codon of the long coding region is sequestered in a region of double-stranded mRNA formed by pairing between the two halves of symmetry B. (iii) IS10-left differs from IS10-right at many nucleotide positions in both the presumptive regulatory region and the long coding region. The available evidence suggests that Tn10 may be older than other analyzed drug-resistance transposons and thus have had more time to accumulate mutational changes.

Base Composition↗
PubMed1981

Three Tn10-associated excision events: relationship to transposition and role of direct and inverted repeats.

We describe three related DNA alterations associated with transposon Tn10: precise excision of Tn10, nearly precise excision of Tn10 and precise excision of the nearly precise excision remnant. DNA sequence analysis shows that each of these alterations results in excision of all or part of the Tn10 element, and each involves specific repeat sequences at or near the ends of the element. Furthermore, all three events are structurally analogous: in each case, excision occurs between two short direct-repeat sequences, with resulting deletion of all intervening material plus one copy of the direct repeat; and in all three cases, the direct repeats involved occur at either end of an inverted repeat. Analysis of mutant Tn10 elements and characterization of bacterial host mutations suggest that all three types of excision events occur by pathways that are fundamentally distinct from the pathway(s) for Tn10-promoted transposition and other DNA rearrangements (deletions and inversions) actively promoted by the element. In addition, precise excision and nearly precise excision appear to occur by very closely related or identical pathways; and several lines of evidence suggest that the 1400 bp inverted repeats at the ends of Tn10 may play a structural role in both of these events. The third excision event appears to occur by yet another pathway.

Base Sequence↗
PubMed1980

Template-independent poly(A) x poly(U) synthesizing activity of different forms of Bacillus subtilis RNA polymerase.

Several, but not all, forms of bacillus subtilis RNA polymerase found in vegetative and sporulating cells can synthesize poly(A) x poly(U) in vitro. The vegetative delta-containing form of RNA polymerase (E delta) has little or no poly(A) x poly(U)-synthesizing activity, whereas RNA polymerase core (E) and sigma-containing core (E delta) both have significant activity. When purified vegetative delta factor was added to core, the core synthetic activity was reduced essentially to that of the vegetative enzyme E delta. When E sigma enzymes from vegetative and sporulating cells were compared for their salt sensitivity, it was found that the sporulation enzyme E sigma retained much more of its activity at 0.1 M KCl than the vegetative enzyme E sigma. Furthermore, when sporulation enzyme E delta 1 was compared with vegetative enzyme E sigma, it was found that the activity of the E sigma 1 form was much more resistant to high KCl concentrations than that of the vegetative E sigma form. These differences in enzyme activity, as affected by salt concentrations, suggest that the conformations of the sporulation E sigma and E delta 1 enzymes are different from that found in vegetative E sigma enzyme. These differences in conformation may be involved in selective gene expression during sporularion.

Bacillus subtilis↗
PubMed1979

Deoxyribonucleic acid sequence common to staphylococcal and streptococcal plasmids which specify erythromycin resistance.

Plasmids from erythromycin-resistant Staphylococcus aureus, Streptococcus sanguis, and Streptococcus faecalis show deoxyribonucleic acid sequence homology. The homologous sequences can be localized to specific restriction endonuclease fragments, which in the case of S. aureus plasmid pI258 involves a single fragment from either EcoRI or HindIII digest known to contain the erythromycin resistance determinant. Complementary ribonucleic acid probes prepared from S. aureus plasmid pI258 and S. sanguis plasmid pAM77 also hybridize to specific fragments in restriction endonuclease digests of deoxyribonucleic acid from erythromycin-resistant Streptococcus progenes and Streptococcus pneumoniae. These studies suggest a common origin for a class of erythromycin resistance determinants in unrelated strains of pathogenic bacteria for which exchange of genetic material has not been demonstrated.

Bacillus subtilis↗
PubMed1977

Reconstitution studies show that rifampicin resistance is determined by the largest polypeptide of Bacillus subtilis RNA polymerase.

A procedure has been developed to separate the subunits of Bacillus subtilis RNA polymerase rapidly and in good yield. The method involved the use of a blue dextran-Sepharose column which bound the beta' subunit. A phosphocellulose column was used to separate the alpha and beta subunits. During purification, the enzyme eluted from the DNA-cellulose column in three separate forms in the order alpha2betabeta'deltaomega1,alpha2betabeta'omega1, and alpha2betabeta'omega1sigma. Subunit reconstitution studies with RNA polymerase subunits from wild type and a rifampicin-resistant mutant indicated that the largest polypeptide was responsible for rifampicin resistance. Thus, this subunit is referred to as beta. The mobility of the subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis cannot be used as the sole criterion for designating the functions of the subunits of RNA polymerase.

Bacillus subtilis↗
PubMed1977

Zinc is associated with the beta subunit of DNA-dependent RNA polymerase of Bacillus subtilis.

The Bacillus subtilis DNA-dependent RNA polymerase holoenzyme and core enzyme each contain approximately two atoms of zinc per molecule. When the dissociated subunits of the enzyme are passed through a blue dextran-Sepharose affinity column, only the beta subunit binds to the column. The total zinc content of the enzyme is tightly bound to the beta subunit. Dialysis studies suggest that the two zinc ions differ in the strength of their association with the beta subunit. The presence of zinc in beta is consistent with several other lines of evidence which indicate that this subunit is dirrectly involved in phosphodiester bond formation. The blue dextran-Sepharose column procedure should be useful in future studies of the dissociation and reassociation of the enzyme since the method is rapid and provides excellent recovery of the beta subunit as well as the alpha and beta' subunits of the RNA polymerase.

Bacillus subtilis↗
PubMed1976

Dihydrodipicolinic acid synthase of Bacillus licheniformis. Quaternary structure, kinetics, and stability in the presence of sodium chloride and substrates.

Dihydrodipicolinic acid synthase (L-aspartate-beta-semialdehyde hydro-lyase (adding pyruvate and cyclising), EC 4.2.1.52) obtained from Bacillus licheniformis was purified to homogeneity. Its molecular weight was 108 000 to 117 500, depending on the concentration of NaCl and substrates present, and it contained four subunits of identical molecular weight (28000). The Km values for pyruvate and L-aspartic semialdehyde were approximately 5.3 Km values for pyruvate and L-aspartic semialdehyde were approximately 5.3 and 2.6 mM, respectively. It was previously shown that pyruvate and a high sodium chloride concentration contributed to the stability of the enzyme. The effect of these substances and the other substrate, L-aspartic semialdehyde, on molecular weight was determined. None of these three substances significantly affected the apparent molecular weight. The effect of sodium chloride, pyruvate, and L-aspartic semialdehyde on enzyme structure was studied by determining the effect of their presence on inactivation of the enzyme by several chemical denaturants and heat. Pyruvate dramatically protected against inactivation by all of the denaturants. Sodium chloride protected against inactivation by sodium dodecyl sulfate, guanidine-HCl, urea, and heat, but somewhat facilitated inactivation by ethanol. L-Aspartic semialdehyde had no significant effect on inactivation by sodium dodecyl sulfate and ethanol; it rendered the enzyme slightly more sensitive to inactivation by guanidine-HCl and urea. The thermal melting curve obtained for the enzyme in the presence of L-aspartic semialdehyde was biphasic. The activity was reduced approximately 50% by heating for 30 min at temperatures between 50 and 80 degrees C. Only by heating at temperatures above 80 degrees C did the inactivation become complete. The partially inactivated enzyme could be reactivated by heating after removal of the L-aspartic semialdehyde. Pyruvate prevented the partial inactivation and facilitated reactivation. The only difference detected between the native enzyme and the partially inactivated form of the enzyme was that the latter had a reduced V. It is known that in other spore-formers, dihydrodipicolinate synthase increases in activity late in sporulation. This increase may be important for normal sporulation to occur. The possibility is discussed that the intracellular pool sizes of pyruvate and L-aspartic semialdehyde might have an influence on the level of dihydrodipicolinate synthase activity, by controlling the amount of partial inactivation of the enzyme that occurs in vivo.

Aldehydes↗