Structure of the HLA-A*0204 antigen,found in South American Indians. Spatial clustering of HLA-A2 subtype polymorphism

The primary structure of the HLA-A2 subtype A^*0204 (isoelectric focusing variant A2.A) has been determined. cDNA encoding this subtype was amplified by the polymerase chain reaction. Four independent full-lenght cDNA clones encoding A^*0204 were analyzed to obtain a consensus sequence for this subtype. A^*0204 differs from A^*0201 by a single nucleotide change of G to T through the coding regions, resulting in an Arg to Met change at position 97. This substitution accounts for the isoelectric focusing pattern of the subtype. The same change occurs in other HLA-A specificities in association with other changes in its vicinity. The absence of additional substitutions in A^*0204 suggests that it could have arisen from A^*0201 by point mutation, and that recurrent mutations may take place during HLA diversification. The spatial location of this change implies that A^*0204 must be a functional variant. Comparison of its sequence with other HLA-A2 subtypes reveals that much of the HLA-A2 subtype polymorphism is generated by variations in four neighboring positions, including position 97, which are located in two adjacent -strands on the floor of the peptide binding site of the molecule.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number X57954. Address correspondence and offprint requests to: J. A. López de Castro.     

Mapping of the structural gene for S-adenosyl homocysteine hydrolase to mouse Chromosome 2, and related sequences to Chromosomes 8 and X

Comparative mapping studies in human and mouse have shown that, to date, human Chromosome (Chr) 20 is completely syntenic with distal mouse Chr 2. The structural locus for S-adenosyl-l-homocysteine hydrolase (EC 3.3.1.1) in human, AHCY, maps to 20 qterq13.1, and we report here that the homologous locus in the mouse, Ahcy, maps to distal mouse Chr 2 with gene order Pcna-Ahcy-Ada. Analysis of 123 progeny of an interspecific backcross between a laboratory stock, AN, and Mus spretus using a rat cDNA probe revealed the presence of at least two other Ahcy-related sequences segregating independently in the mouse genome. One, Ahcy-rs1, was mapped to Chr 8 in the BXH recombinant inbred strains, and the other, Ahcy-rs2, shows a pattern of inheritance consistent with X-linkage.     

Lectin binding sites on head structures of the spermatid and spermatozoon of the mosquito Culex quinquefasciatus (Diptera,Culicidae)

The presence of intranuclear and acrosomal lectin binding sites in spermatids and spermatozoa of the mosquito Culex quinquefasciatus was analysed. Direct and indirect lectin-gold techniques were used on LR White-embedded cells. The nuclear compartment was the structure most intensely labelled. Early spermatid nucleus showed moderate labelling for peanut agglutinin (PNA), Griffonia simplicifolia IB4 (GS-IB4) and Ricinus communis agglutinin (RCA), and light labelling for the other lectins tested. The sperm nucleus was intensely labelled by all lectins. The acrosome, an enzyme-containing structure, was labelled by some lectins. The anterior acrosomal region was labelled by PNA, while the proximal acrosomal region was labelled by PNA and G. simplicifolia II (GS II) lectins, and showed the presence of fucose residues with the use of Ulex europaeus I (UEA-I) lectin. The spermatozoa stored in the spermatheca showed the same pattern of labelling as that observed in spermatozoa localized in testis and seminal vesicles for all lectins tested. Carbohydrate residues in the nuclear compartment may be involved with the process of chromatin condensation. In the acrosomal region these residues may play a role in the process of spermoocyte interaction.     

Molecular and symbiotic characterization of exopolysaccharide-deficient mutants of Rhizobium tropici strain CIAT899

We studied the symbiotic behaviour of 20 independent Tn5 mutants of Rhizobium tropici strain CIAT899 that were deficient in exopolysaccharide (EPS) production. The mutants produced non-mucoid colonies, were motile, grew in broth cultures at rates similar to those of the parent, and produced significantly less EPS than did CIAT899 in broth culture. A genomic library of strain CIAT899, constructed in pLA2917, was mobilized into all of the mutants, and cosmids that restored EPS production were identified. EcoRI restriction digests of the cosmids revealed nine unique inserts. Mutant complementation and hybridization analysis showed that the mutations affecting EPS production fell into six functional and physical linkage groups. On bean, the mutants were as efficient in nodulation and as effective in acetylene reduction as strain CIAT899, induced a severe interveinal chlorosis, and all but one were less competitive than CIAT899. On siratro, CIAT899 induced nodules that were ineffective in acetylene reduction, whereas the EPS-deficient mutants induced effective nodules. Microscopic examination of thin sections showed that nodules from both siratro and bean plants inoculated with either CIAT899 or an EPS-deficient mutant contained infected cells. These data indicate that EPS is not required for normal nodulation of bean by R. tropici, that it may contribute to competitiveness of R. tropici on bean, and that the loss of EPS production is accompanied by acquisition of the ability to reduce acetylene on siratro.     

gltF, a member of the gltBDF operon of Escherichia coli, is involved in nitrogen-regulated gene expression

We report here the construction and analysis of insertional mutations in each of the three genes of the gltBDF operon and the nucleotide sequence of the region downstream from gltD. Two open reading frames were identified, the first of which corresponds to gltF. The gltB and gltD genes code for the large and small subunits, respectively, of the enzyme glutamate synthase (GOGAT). gltF codes for a protein, with a molecular mass of 26,350 Da, which is required for Ntr induction. Histidase synthesis was determined as a measure of Ntr function. First, insertions in gltB, gltD or gltF all prevent Ntr induction. Second, complementation analysis indicates that high-level expression of both the gltD and gltF genes is required for the induction of the Ntr enzymes under nitrogen-limiting conditions, indicating that the phenotype of the gltB insertion probably results from polarity on gltD and gltF. Third, glutamate-dependent repression of the glt operon appears to be mediated by the product of the gltF gene. Thus, the gltBDF operon of Escherichia coli is involved in induction of the so-called Ntr enzymes in response to nitrogen deprivation, as well as in glutamate biosynthesis.     

gltBDF operon of Escherichia coli.

A 2.0-kilobase DNA fragment carrying antibiotic resistance markers was inserted into the gltB gene of Escherichia coli previously cloned in a multicopy plasmid. Replacement of the chromosomal gltB+ gene by the gltB225::omega mutation led to cells unable to synthesize glutamate synthase, utilize growth rate-limiting nitrogen sources, or derepress their glutamine synthetase. The existence of a gltBDF operon encoding the large (gltB) and small (gltD) subunits of glutamate synthase and a regulatory peptide (gltF) at 69 min of the E. coli linkage map was deduced from complementation analysis. A plasmid carrying the entire gltB+D+F+ operon complemented cells for all three of the mutant phenotypes associated with the polar gltB225::omega mutation in the chromosome. By contrast, plasmids carrying gltB+ only complemented cells for glutamate synthase activity. A major tricistronic mRNA molecule was detected from Northern (RNA blot) DNA-RNA hybridization experiments with DNA probes containing single genes of the operon. A 30,200-dalton polypeptide was identified as the gltF product, the lack of which was responsible for the inability of cells to use nitrogen-limiting sources associated with gltB225::omega.     

Phospholipid species containing long and very long polyenoic fatty acids remain with rhodopsin after hexane extraction of photoreceptor membranes

About one-fourth the phosphatidylcholines (PCs) from bovine disk photoreceptor membranes contain very long chain (24-36 carbons) polyunsaturated (4, 5, and 6 double bonds) fatty acids of the n-3 and n-6 series (VLCPUFA). Such fatty acids, exclusively occurring in dipolyunsaturated species, are esterified to the sn-1 position of their glycerol backbone, docosahexaenoate being the major fatty acid at sn-2. Chromatographically, such PCs display a weakly polar character relative to other species, ascribable to their exceedingly large number of carbons. After hexane extraction of lyophilized disks, PC is the major component of the fraction of lipids that remains associated with rhodopsin, followed by phosphatidylserine, while a large proportion of the phosphatidylethanolamine is removed. The fatty acid composition of the hexane-removable and protein-bound lipid fractions markedly differs, the latter being enriched in lipid species containing long-chain and very long chain polyenes. This is observed for all lipid classes except free fatty acids. VLCPUFA-containing PCs are the most highly concentrated species in the rhodopsin-associated lipid fraction. The very long chain polyenes these PCs have at sn-1 may account for their resistance to being separated from the protein. It is hypothesized that their unusually long polyenoic fatty acids could be well suited to partially surround alpha-helical segments of rhodopsin.     

Sulfated glycosaminoglycans synthesized by human smooth muscle cells isolated from different organs

The sulfated glycosaminoglycans synthesized by human smooth muscle cells isolated from different organs were identified on the basis of electrophoretic mobility, enzymatic degradation with specific mucopolysaccharidases and by the type of degradation products formed. The results obtained indicated that chondroitin sulfate and heparan sulfate were the main glycosaminoglycans found, that most of the labeled glycosaminoglycans were found in the pericellular pool, and that no marked differences were observed in the sulfated glycosaminoglycan composition of the smooth muscle cells obtained from different organs. 'Liver connective tissue cells', isolated from pathological livers (which had been shown to possess biochemical and physiological features typical of smooth muscle cells) showed a pattern of glycosaminoglycan synthesis similar to that of the smooth muscle cells.     

Repair of chemical carcinogen-induced damage in DNA of human lymphocytes and lymphoid cell lines--studies of the kinetics of removal of O6-methylguanine and 3-methyladenine

The removal of O6-methylguanine by human lymphoid cells corresponded, with certain assumptions, to a second-order chemical reaction in any given cell. There was a spectrum of proficiency in this respect for a considerable number of cells originating from different individuals and it was found that patients with diseases associated with autoimmunity tended to fall into the less proficient groups. E-B virus-induced lymphoid cell lines, derived from proficient, but not relatively deficient, peripheral blood lymphocytes, always (in 9/9 cases) reflected the level of proficiency of the donor lymphocytes with respect to removal of O6-methylguanine. Thus while proficient lymphocytes always produced proficient cell lines, deficient lymphocytes, in 3/8 cases, gave rise to more proficient cell lines. No evidence was found that groups of individuals exist who lack ability to remove 3-methyladenine from DNA, either from their blood lymphocytes or derived lymphoid cell lines.