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A Sutton

Publications and source records attributed to A Sutton.

At least 73 records · Page 4Linked to original sources
PubMed1985

Signals for transcription initiation and termination in the Saccharomyces cerevisiae plasmid 2 micron circle.

By S1 nuclease protection experiments and primer extension analysis, we determined precisely the cap and polyadenylation sites of transcripts from the four genes of the yeast 2 micron circle plasmid, as well as those of other plasmid transcripts of unknown function. In addition, we used deletion analysis to identify sequences necessary for polyadenylation in plasmid transcripts. Our results indicate that plasmid genes constitute independent transcription units and that plasmid mRNAs are not derived by extensive processing of precursor transcripts. In addition, we found that the D coding region of 2 micron circle is precisely encompassed by a polyadenylated transcript, suggesting that this coding region constitutes a functional plasmid gene. Our identification of the position of plasmid polyadenylation sites and of sequences necessary for polyadenylation provides support for a tripartite signal for polyadenylation as proposed by Zaret and Sherman (K.S. Zaret and F. Sherman, Cell 28:563-573, 1982). Finally, these data highlight salient features of the transcriptional regulatory circuitry that underlies the control of plasmid maintenance in the cell.

Base Sequence↗
PubMed1985

Properties of REP3: a cis-acting locus required for stable propagation of the Saccharomyces cerevisiae plasmid 2 microns circle.

Stable propagation of the yeast plasmid 2 microns requires an origin of replication, a cis-active locus designated REP3, and two plasmid-encoded proteins which are the products of the REP1 and REP2 genes. The three REP loci appear to constitute a partitioning system, ensuring equal distribution of plasmid molecules to mother and daughter cells after mitosis. We have localized the REP3 site completely within a segment of five-and-one-half direct tandem repeats of a 62-base-pair unit, bordered by HpaI and AvaI restriction sites within the large unique region of the 2 microns genome. In addition, we find that the repeated elements are functionally distinct. Only a subset of the repeats is necessary to promote full partitioning activity. The other repeats appear to promote plasmid transcription. These results are discussed in the context of a model of plasmid copy control involving titration of a plasmid-specific protein by the repeated elements within REP3.

Base Sequence↗
PubMed1984

Serum antibody responses of juvenile and infant rhesus monkeys injected with Haemophilus influenzae type b and pneumococcus type 6A capsular polysaccharide-protein conjugates.

Juvenile and infant rhesus monkeys were injected subcutaneously with saline solutions of Haemophilus influenzae type b (Hib) and pneumococcus type 6A (Pn6A) capsular polysaccharides conjugated to either tetanus toxoid (TT), horseshoe crab hemocyanin, or cholera toxin (CT), and the antibody responses of the monkeys to both bacterial components were measured. All three Hib conjugates were immunogenic and elicited booster responses; their comparative immunogenicity was Hib-CT greater than Hib-TT greater than Hib-horseshoe crab hemocyanin. Hib alone did not elicit antibodies in the juveniles. Juveniles responded earlier and with higher levels of antibodies than did infants. TT, as well as diphtheria-tetanus toxoids-pertussis vaccine adsorbed injected concurrently at a separate site, increased both Hib and TT antibody responses in juveniles (P less than 0.05). Concurrent injection of 5 Lf of fluid TT with a nonimmunogenic 5-micrograms dose in infants elicited levels of Hib antibodies comparable to those elicited by 50 micrograms of Hib-TT. Hib antibodies elicited by the conjugates remained at protective levels in both juveniles and infants 2 months after the last injection, were bactericidal, and conferred passive immunity against bacteremia in infant rats. Passive immunization of juveniles with tetanus immune globulin before each injection of Hib-TT did not suppress Hib antibodies. Hib-TT and Hib-CT elicited increases of Hib antibodies of the immunoglobulin M and G isotypes in the infants. The Pn6A-TT conjugate was considerably less immunogenic than the Hib-TT conjugate; only a few of the juveniles or infants responded with protective levels of Pn6A antibodies. Pn6A antibodies from responders conferred protection in mice against intraperitoneal challenge with Pn6A organisms. TT antibodies were elicited in both juvenile and infant animals after one injection of 50 micrograms of Hib-TT and in the infants injected with 5 micrograms of Hib-TT plus 5 Lf of TT; 5 micrograms of Hib-TT and Pn6A-TT in combination alone did not elicit TT antibodies. Hib-CT elicited CT antibodies in both juveniles and infants.

Adjuvants, Immunologic↗
PubMed1983

Class-specific antibody response to Haemophilus influenzae type b capsular polysaccharide vaccine.

Bacteremic infections caused by H influenzae type b have their peak incidence in children around one year of age, at a time when the serum antibody response to the capsular polysaccharide is very low [1, 3]. The capsular polysaccharide vaccine has in fact been shown to be efficacious in preventing the disease above but not below the age of 18 months [1]. Preliminary observations supported the notion that slow maturation of cells that synthesize antibody to H influenzae type b altered immunoglobulin class distribution of the response and resulted specifically in a defective IgG antibody response [1]. We have used a solid-phase ELISA to measure the class-specific antibody responses in 14 infants and children vaccinated with the capsular polysaccharide vaccine. We found that, among the infants who were less than 18 months old and whose antibody response was generally low, the response was only IgG-specific in two children and only IgM-specific in two others. In one child an IgG and IgM response was detected, and in two no response was detected. Among the older children, all children showed an increase in IgG antibody; only two also had an increase of IgM antibody. IgA antibodies were not detectable in any preimmune sera. The first IgA antibody responses were seen at the age of 15 months but were common thereafter. These data do not indicate a specific defect of IgG-class antibodies in the antibody response to the capsular polysaccharide vaccine.

Antibodies, Bacterial↗
PubMed1983

Six widespread bacterial clones among Escherichia coli K1 isolates.

Variable properties among Escherichia coli isolates include serotype, electrophoretic migration of major outer membrane proteins, metabolic properties, production of hemolysin or colicin or both, and plasmid content. These characteristics were compared in E. coli strains of capsular types K1, K5, K92, and K100 and in non-encapsulated isolates. The 234 bacterial strains from the United States and Europe which we studied had been isolated from healthy or diseased individuals recently or as long ago as 1941. Regardless of source, most O7:K1, O16:K1, and O75:K100 isolates could be assigned to three unique, serotype-specific groups, which were interpreted as representing three bacterial clones. Two bacterial (sub)clones each were discerned among the O18:K1 and O18:K5 isolates, and two further, distinct clones were discerned among the O1:K1 isolates. The implications of these results for epidemiological analyses and for virulence are discussed.

Cell Membrane↗
PubMed1982

Cloning and expression of the ilvB gene of Escherichia coli K-12.

A plasmid containing the ilvB operon, which codes for acetohydroxy acid synthase I of Escherichia coli K-12, was isolated using a ligated mixture of DNA from plasmid pBR322 and FilvB4 treated with endonuclease SalI. A shortened derivative of this plasmid was isolated by cloning a 3.4 kb bacterial fragment into plasmid pKEN005 to yield plasmid pTCN12. The orientation of the ilvB operon relative to plasmid genes was determined by restriction enzyme mapping. Measurement of the level of the product of the ilvB gene, acetohydroxy acid synthase I, indicated that plasmid pTCN12 contained a functional ilvB promoter and control region. The DNA frm this plasmid was used as a probe to show that the rate of synthesis of ilvB mRNA was proportional to the levels of acetohydroxy acid synthase I.

Acetolactate Synthase↗
PubMed1982

The mucosal immune response in the neonate.

Human infants are relatively deficient in the IgA system defending mucosal membranes, but are provided via the maternal milk with considerable amounts of SIgA directed against microbes and food antigens to which both mother and infant are exposed. It is possible that serum antibodies may support the mucosal defense as do the lactoferrin, lysozyme and other defense factors present in the milk.

Animals↗
PubMed1982

Differential complement resistance mediates virulence of Haemophilus influenzae type b.

Studies were undertaken to gain insight into the virulence of type b in contrast to the other Haemophilus influenzae capsular types. A relationship was found between the comparative virulence of H. influenzae types in humans and their resistance to the bactericidal effect of antibody-free complement. Type b was most resistant to the bactericidal effect of complement. The other types could be divided into three groups based upon their susceptibility to complement; this grouping was also related to their structural similarities. No association between virulence and either the biotype, source of isolate, in vitro association with peripheral polymorphonuclear leukocytes, or the total amount of capsular polysaccharide was found. However, among the type b strains, higher levels of cell-associated polysaccharide were associated with increased resistance to complement. The relative virulence of the six H. influenzae types in the infant rat model was generally similar to that in humans. After intraperitoneal challenge, type b and type a strains had the lowest 50% effective doses for bacteremia, removed by several logs from the values of the other types. By intranasal challenge, type b strains produced higher rates and levels of bacteremia than did type a strains. High levels of natural bactericidal antibodies to types c and e were found in adult female rats; this finding alone could not account for the differences in virulence among the H. influenzae types in the infant rat model. We propose that the virulence of type b strains is due to their greater resistance to the bactericidal activity of serum complement alone. Resistance to type b disease requires serum antibody to induce the complement-mediated reaction.

Animals↗
PubMed1982

Mutations in genes cpxA and cpxB of Escherichia coli K-12 cause a defect in acetohydroxyacid synthase I function in vivo.

Mutations in Escherichia coli genes cpxA and cpxB together cause a temperature-sensitive defect in isoleucine and valine syntheses that is related specifically to acetohydroxyacid synthase I. This enzyme catalyzes the first pair of homologous reactions required for the synthesis of these two amino acids. At both permissive and nonpermissive temperatures, mutant cells containing ilvB (the structural gene for acetohydroxyacid synthase I) cloned in a derivative of plasmid pBR322 synthesized comparable amounts of ilvB mRNA and contained several times the enzyme activity normally required to sustain exponential growth, yet these cells remained temperature sensitive for growth in the absence of isoleucine and valine. These observations suggest that the primary effect of the cpx mutations is to block enzyme function in vivo. The enzyme was unstable in mutant cells at growth temperatures above 37 degrees C, but this instability appeared to be a secondary effect on the cpx mutations.

Acetolactate Synthase↗
PubMed1982

The requirement of specific anticapsular IgG for killing of Haemophilus influenzae by the alternative pathway of complement activation.

Employing an in vitro bactericidal assay using C4-deficient guinea pig serum (C4D-GPS), we evaluated the ability of the alternative pathway to kill a number of strains of encapsulated and unencapsulated Haemophilus influenzae (HI). Complement activation occurred, but no bactericidal activity was observed against any of the tested HI in nonimmune C4D-GPS without detectable antibody to HI-type b (HIb) capsular polysaccharide (HIb-ps). In the presence of high-titered human anti- (type b) IgG, C4D-GPS killed the encapsulated strains. Restoration of classical pathway activity by the addition of purified C4 to C4D-GPS, which contained antibody to somatic antigens, resulted in killing of some strains of both encapsulated and unencapsulated HI. In nonimmune C4D-GPS the alternative pathway is activated but does not mediate bactericidal activity. The addition of specific high-titered anticapsular IgG results in killing of encapsulated HI by the alternative pathway in guinea pig serum.

Animals↗
PubMed1980

Preparation, characterization, and immunogenicity of Haemophilus influenzae type b polysaccharide-protein conjugates.

A method is presented for covalently bonding Haemophilus influenzae type b capsular polysaccharide (HIB Ps) to several proteins. The method is efficient and relies upon the use of adipic dihydrazide as a spacer between the capsular polysaccharide and the carrier protein. In contrast to the poor immunogenicity of the purified HIB Ps in mice and rabbits, the HIB Ps-protein conjugates induced serum anti-type b antibodies having bactericidal activity at levels shown to be protective in humans when low doses were injected subcutaneously in a saline solution. The antibody response in mice was related to the dose of the conjugates, increased with the number of injections, and could be primed by the previous injection of the carrier protein. The HIB Ps-protein conjugates were immunogenic in three different mouse strains. The importance of the carrier molecule for the enhanced immunogenicity of the HIB Ps-protein conjugates was shown by the failure of HIB Ps hybrids prepared with either the homologous polysaccharide or pneumococcus type 3 polysaccharide to induce antibodie in mice. Rabbits injected with the HIB Ps-protein conjugates emulsified in Freund's adjuvant produced high levels of serum anti-type b antibodies which induced a bactericidal effect upon H. influenzae type b organisms. It is proposed that the HIB Ps component of the polysaccharide protein conjugates has been converted to a thymic-dependent immunogen. This method may be used to prepare protein-polysaccharide conjugates with HIB Ps and other polysaccharides to be considered for human use.

Animals↗
PubMed1980

Regulation of cyclic AMP of the ilvB-encoded biosynthetic acetohydroxy acid synthase in Escherichia coli K-12.

The biosynthetic acetohydroxy acid synthase activities of E. coli K 12 are encoded by three genetic loci namely, ilvB (acetohydroxy acid synthase I), ilvG (acetohydroxy acid synthase II) and ilvHI (acetohydroxy acid synthase III). The previously reported involvement of cyclic AMP in the regulation of the biosynthetic acetohydroxy acid synthase isozymes in E. coli K-12 was found to be due to the effect of this nucleotide on the expression of ilvB. Cyclic AMP had no effect on acetohydroxy acid synthase activity in strains lacking wild-type ilvB activity but containing the remaining isozymes. Very little activity of acetohydroxy acid synthase coded for by ilvV was found when ppGpp and cyclic AMP were severely limited. Addition of cyclic AMP under these conditions increased ilvB expression 24-fold. The data suggest that in addition to multivalent repression and ppGpp, cyclic AMP plays a major role in the regulation of the ilvB biosynthetic operon.

Acetolactate Synthase↗
PubMed1980

Comparative analysis of plasmids and some metabolic characteristics of Escherichia coli K1 from diseased and healthy individuals.

All 62 Escherichia coli strains possessing the K1 capsular polysaccharide contained plasmid deoxyribonucleic acid, and most (51 of 62) had multiple plasmid species. The incidence of hemolysins, colicins, hemagglutinins for human erythrocytes, and plasmids did not differ among K1 strains isolated from the cerebrospinal fluids of neonates with meningitis or among those strains isolated from the stools of healthy individuals of all ages. There was an association between E. coli serotype and the distribution of plasmids, hemolysins, and colicins among the K1 strains. A common plasmid of about 65 megadaltons was found in all of the O18 serotypes; the similarity of these plasmids was confirmed by analysis with the restriction endonuclease EcoRI. Plasmids of similar molecular weight were also present in E. coli strains of the O7:K1 and O75:K100 serogroups. These data are consistent with the hypothesis that E. coli strains of the same serotype may be descendents of a single bacterial clone.

Adult↗