Genetic mapping of Vibrio cholerae enterotoxin structural genes

The structural genes which constitute the cholera toxin operon, ctxAB, were genetically mapped in the Vibrio cholerae El Tor strain RV79. This strain of V. cholerae contains two copies of the ctx operon located on a 7-kilobase-pair tandemly duplicated region. We began by isolating a vibriophage VcA1 insertion mutation in one of the two ctxA genes located in this region. The mutant carrying this ctxA::VcA1 insertion, DC24, was converted to a VcA1-facilitated donor by introduction of the conjugal plasmid pSJ15, which carries an inserted copy of a defective VcA1-like prophage. The donor characteristics of DC24(pSJ15) indicated that the ctxA::VcA1 insertion mutation was near the trp region of the V. cholerae chromosome. Subsequent RV79 three-factor crosses were performed between VcA1-facilitated donors and recipient strains carrying one of two structural gene mutations in ctx, either delta ctxA23P Kmr or delta ctx-7922. The former was constructed by an in vivo marker exchange procedure and could be scored either by its kanamycin resistance phenotype or by its lack of DNA sequences homologous to the ctxA region. The delta ctx-7922 mutation is a total deletion of both ctx copies of strain RV79. The three-factor cross data strongly suggest that the two ctx loci of RV79 map between the nal and his genes of V. cholerae in the trp nal his linkage group. Physical analysis and heterologous crosses between an RV79 El Tor donor and a 569B classical recipient indicates that one of the two 569B ctx operon copies maps in the same region as the RV79 ctx loci (i.e., linked to nal). Together with previously published observations, these data show that the ctx structural genes are not closely linked to other genes known to affect toxin production in V. cholerae.     

Reactions of sugar nitro-alkenes with acetoacetic esters

3,4,5,6,7-Penta-O-acetyl-1,2-dideoxy-1-nitro-d-gluco-hept-1-enitol reacts with methyl acetoacetate in an unusual Michael reaction, giving the normal adduct (6), and a bicyclic derivative (9) that arises from quasi-dimerization of the former when a high concentration of the base is used. Acetylation of compound 9 gives the hydroxylamine O-acetate (10).From the reactions of 3,4,5,6,7-penta-O-acetyl-1,2-dideoxy-1-nitro-d-galacto-hept-1-enitol with ethyl and tert-butyl acetoacetate, the normal adducts (7 and 8) were isolated. The structures of compounds 6 to 10 were established on the basis of their spectral properties (u.v., i.r., mass, and ¹H- and ¹³C-n.m.r.)     

Reaction of ethyl 2-amino-4,6-O-benzylidene-2-deoxy-d-gluconate with acetylenic esters

Ethyl 2-amino-4,6-O-benzylidene-2-deoxy-d-gluconate adds to acetylenic esters to give sugar enaminones. The following acetylene derivatives have been employed: methyl propiolate, ethyl phenylpropiolate, and dimethyl acetylenedicarboxylate (6). With compound 6, the reaction leads to a mixture of the expected enaminone and the isomeric oxazolidine derivative. The structures and configurations of the new compounds were studied by spectroscopic and chemical methods.     

Variability and conformation of HLA class I antigens: a predictive approach to the spatial arrangement of polymorphic regions

Comparison of available sequences of HLA-A and HLA-B antigens shows that variable positions are predominantly localized in four segments spanning residues 63-85, 105-116, 138-156, and 177-194. The fourth segment is unique in that it contains no differences between antigens of the same locus. Secondary folding of HLA heavy chain was estimated by three independent predictive methods and areas of defined structure were correlated with the distribution of local hydrophobicity to outline putative internal and external portions. The three analyses each independently predict a high probability for beta structure in the alpha 1, alpha 2, and alpha 3 domains. A single alpha-helix is predicted within residues 146-160, a segment of likely importance in cytotoxic T cell recognition and graft rejection. Substitutions within this segment are spatially related by the helical turn. Variable residues usually lie in areas of high local hydrophilicity, and therefore they are probably on the surface of the molecule. The model predicts that they are frequently located in beta strands, beta-turns, or the above-mentioned alpha-helix, so that most substitutions would be accommodated within rigid frameworks that may impose structural constraints to variability. The secondary structure of alpha 1, alpha 2, and alpha 3 domains presents some analogies that suggest that they might share common features in their tertiary folding. The predicted structure of alpha 3 is strongly reminiscent of that of immunoglobulin constant domains. Possible arrangements of elements of secondary structure are discussed, as an attempt to situating the polymorphic regions of HLA class I antigens in a spatial context.     

Physical structure and genetic expression of the sulfonamide-resistance plasmid pLS80 and its derivatives in Streptococcus pneumoniae and Bacillus subtilis

Summary The 10-kb chromosomal fragment of Streptococus pneumoniae cloned in pLS80 contains the sul-d allele of the pneumococcal gene for dihydropteroate synthase. As a single copy in the chromosome this allele confers resistance to sulfanilamide at 0.2 mg/ml; in the multicopy plasmid it confers resistance to 2.0 mg/ml. The sul-d mutation was mapped by restriction analysis to a 0.4-kb region. By the mechanism of chromosomal facilitation, in which the chromosome restores information to an entering plasmid fragment, a BamHI fragment missing the sul-d region of pLS80 established the full-sized plasmid, but with the sul-s allele of the recipient chromosome.A spontaneous deletion beginning 1.5 kb to the right of the sul-d mutation prevented gene function, possibly by removing a promoter. This region could be restored by chromosomal facilitation and be demonstrated in the plasmid by selection for sulfonamide resistance. Under selection for a vector marker, tetracycline resistance, only the deleted plasmid was detectable, apparently as a result of plasmid segregation and the advantageous growth rates of cells with smaller plasmids. When such cells were selected for sulfonamide resistance, the deleted region returned to the plasmid, presumably by equilibration between the chromosome and the plasmid pool, to give a low frequency (10^-3) of cells resistant to sulfanilamide at 2.0 mg/ml. Models for the mechanisms of chromosomal facilitation and equilibration are proposed.Several derivatives of pLS80 could be transferred to Bacillus subtilis, where they conferred resistance to sulfanil-amide at 2 mg/ml, thereby demonstrating cross-species expression of the pneumococcal gene. Transfer of the plasmids to B. subtilis gave rise to large deletions to the left of the sul-d marker, but these deletions did not interfere with the sul-d gene function. Restriction maps of pLS80 and its variously deleted derivatives are presented.     

Differences in glycerolipid synthesis and insulin regulation in rat hepatocytes and adipocytes

Glycerolipid synthesis was studied by determining radioactive incorporation from either [1-14C] acetate or [U-14C] palmitate. Glycerolipid synthesis in adipocytes, mainly from exogenous palmitate, was preferentially directed to the formation of triacylglycerols, whereas in hepatocytes triacylglycerols and phospholipids were synthesized at similar rates. Insulin stimulated glycerolipid synthesis from acetate in both types of cells, being triacylglycerols more significantly increased than phospholipids. The most relevant difference was the finding that in adipocytes insulin strongly stimulated the formation of diglycerides, apparently from phosphatidate, whereas in hepatocytes insulin only slightly increased diglyceride levels. A possible role of diacylglycerol in insulin action in adipocytes, but not in hepatocytes, is also discussed.     

Regulatory interaction of photosynthetic nitrate utilization and carbon dioxide fixation in the cyanobacterium Anacystis nidulans

The rate of photosynthetic nitrate utilization in Anacystis nidulans is strongly influenced by the availability of carbon dioxide. This dependence can be relieved by inhibiting the metabolism of the ammonium derived from nitrate reduction. Nitrate uptake seems to be modulated through a sensitive regulatory system integrating the photosynthetic metabolism of carbon and nitrogen, with CO₂ fixation products antagonizing the inhibitory effect of ammonium derivatives.     

Desangeloylshairidin,X-ray structure determination of a sesquiterpene lactone from Guillonea scabra

The structure and absolute configuration of desangeloylshairidin, a guaianolide isolated from Guillonea scabra, have been established by X-ray diffraction analysis. No conformational change was observed in its seven-membered ring between the crystal and deuterochloroform solution states.     

Heritable fragile sites on human chromosomes. X. New folate-sensitive fragile sites: 6p23, 9p21, 9q32, and 11q23

Four new folate-sensitive fragile sites are documented at 6p23, 9p21, 9q32, and 11q23. These have all been shown to be heritable except for the one at 9p21, which has been seen only in a single individual. As with the other autosomal fragile sites, these appear to be innocuous in heterozygotes.