Effects of non-MHC loci on resistance to retrovirus-induced immunodeficiency in mice

Mice of certain strains are highly sensitive to development of a severe immunodeficiency disease following inoculation as adults with LP-BM5 murine leukemia viruses (MuLV) whereas others are extremely resistant. These strain-dependent differences in response to infection have been shown to be genetically determined with resistance to disease being, in general, associated with homozygosity for Fv-1 ⁿand H-2 haplotypes a and d and sensitivity with homozygosity for Fv-1 ^band other H-2 haplotypes including b, s, and q. The Fv-1 ^b, H-2 ^rstrain RIIIS/J (RIIIS) was found to be highly resistant to disease even though B10.RIII(71NS)/J (B10.RIII), also H-2 ^r, was very sensitive, thus excluding a role for H-2 in the resistance of RIIIS. The characteristics of RIIIS resistance were evaluated in studies of infected (B10.RIII×RIIIS) F₁, F₂ and reciprocal backcross mice. Resistance to disease was shown to be semidominant and determined by more than one gene, although a preponderant influence of a single gene was suggested. Studies of segregating populations showed that resistance was not associated with or linked to polymorphisms of the V _\complex or genes in proximity to the Emv-2 locus on chromosome 8. However, there was almost complete concordance between absence of disease in infected mice and inhibition of ecotropic virus spread. These results demonstrate that genes other than Fv-1 or H-2 can profoundly influence the development of retrovirus-induced immunodeficiency and replication of ecotropic viruses.Abbreviations MuLV murine leukemia virus - MCF mink cell focus-inducing MuLV - B6 C57BL/6 - BM5d the defective virus in LP-BM5 MuLV - MAIDS murine acquired immunodeficiency syndrome - RIIIS RIIIS/J - B10.RIII B10.RIII (71NS)/J - MLR mixed lymphocyte reaction - FACS fluorescence activated cell sorter     

Characterization of matrix domains of the hamster acrosome

In this study we describe the purification and the structural and biochemical properties of a detergent-stable complex of the hamster sperm acrosome. This complex consists of two distinct acrosomal matrix domains and a layer of electron-dense material, termed the acrosomal lamina, derived from the luminal surface of the outer acrosomal membrane. This complex has been isolated by centrifugation of detergent-extracted sperm suspensions on Percoll density gradients. The complex contains two major polypeptides of Mr 29,000 and Mr 22,000 and minor polypeptides of Mr 64,000-62,000, 56,000 and 35,000. Gelatin-containing sodium dodecyl sulfate-polyacrylamide gels demonstrate that bands of proteinase activity are not the major polypeptide components of the complex. These data demonstrate that the matrix of the acrosome is compartmentalized into domains of differing structural properties that occupy specific locations in the intact acrosome and that matrix components are physically associated with the outer acrosomal membrane. These data indicate that a structural framework is present within the acrosome and we speculate that it may be involved in sequestering hydrolases into specific spatial domains and could affect the temporal release of activity of selected hydrolases during the acrosome reaction.     

Phylogeny of the 5S ribosomal RNA fromSynechococcus lividus II: The cyanobacterial/chloroplast 5S RNAs form a common structural class

Summary The complete nucleotide sequence of the 5S ribosomal RNA from the cyanobacteriumSynechococcus lividus II has been determined. The sequence is 5-UGCCUAGUGUUUAUGGCGCG-GUGGAACCACGCUGAUCCAUCCCGAACUC-AGAGGUGAAACAUCGCAGCGGUGAAGAU-AGUUGGAGGGUAGCCUCCUGCAAAAAUA-GCUCAAUGCUAGGCA_OH-3. This 5S RNA has the cyanobacterial- and chloroplast-specific nucleotide insertion between positions 30 and 31 (using the numbering system of the generalized eubacterial 5S RNA) and the chloroplast-specific nucleotide-deletion signature between positions 34 and 39. The 5S RNA ofS. lividus II has 27 base differences compared with the 5S RNA of the related strainS. lividus III. This large difference may reflect an ancient divergence between these two organisms. The electrophoretic mobilities on nondenaturing polyacrylamide gels of renatured 5S RNAs fromS. lividus II,S. lividus III, and spinach chloroplasts are identical, but differ considerably from that ofEscherichia coli 5S RNA. This most likely reflects differences in higher-order structure between the 5S RNA ofE. coli and these cyanobacterial and chloroplast 5S RNAs.     

Structure of membrane domains and matrix components of the bovine acrosome

The acrosomal membrane system of bovine spermatozoa was examined by thin-section, freeze-fracture, surface-replica, and negative staining techniques in order to identify structural differentiations of specific acrosomal membrane domains. The outer acrosomal membrane of the apical and principal segments is characterized by a prominent electron-dense complex associated with its luminal face and a random intramembranous particle distribution. In the equatorial segment, the two-dimensional organization of bridging elements extending between the outer and inner acrosomal membrane was determined and correlated to freeze-fracture images. The inner acrosomal membrane lacked the electron-dense assembly noted on the outer acrosomal membrane and in freeze-fracture it appears crystalline. Further studies identified the distribution of the electron-dense subacrosomal material in the space between the inner acrosomal membrane and outer nuclear membrane. Finally, new observations on the structural organization of the acrosomal matrix are presented.     

Temperature-Sensitive Lethal Mutations on Yeast Chromosome I Appear to Define Only a Small Number of Genes

A method was developed for isolating large numbers of mutations on chromosome I of the yeast Saccharomyces cerevisiae. A strain monosomic for chromosome I (i.e., haploid for chromosome I and diploid for all other chromosomes) was mutagenized with either ethyl methanesulfonate or N-methyl-N'-nitro-N -nitrosoguanidine and screened for temperature-sensitive (Ts^- ) mutants capable of growth on rich, glucose-containing medium at 25° but not at 37°. Recessive mutations induced on chromosome I are expressed, whereas those on the diploid chromosomes are usually not expressed because of the presence of wild-type alleles on the homologous chromosomes. Dominant ts mutations on all chromosomes should also be expressed, but these appeared rarely. — Of the 41 ts mutations analyzed, 32 mapped on chromosome I. These 32 mutations fell into only three complementation groups, which proved to be the previously described genes CDC15, CDC24 and PYK1 (or CDC19). We recovered 16 or 17 independent mutations in CDC15, 12 independent mutations in CDC24 and three independent mutations in PYK1. A fourth gene on chromosome I, MAK16, is known to be capable of giving rise to a ts-lethal allele, but we recovered no mutations in this gene. The remaining nine mutations isolated using the monosomic strain appeared not to map on chromosome I and were apparently expressed in the original mutants because they had become homozygous or hemizygous by mitotic recombination or chromosome loss. — The available information about the size of chromosome I suggests that it should contain approximately 60–100 genes. However, our isolation in the monosomic strain of multiple, independent alleles of just three genes suggests that only a small proportion of the genes on chromosome I is easily mutable to give a Ts^--lethal phenotype. — During these studies, we located CDC24 on chromosome I and determined that it is centromere distal to PYK1 on the left arm of the chromosome.     

Recognition of super-secondary structure in proteins

A procedure to recognize super-secondary structure in protein sequences is described. An idealized template, derived from known super-secondary structures, is used to locate probable sites by matching with secondary structure probability profiles. We applied the method to the identification of βαβ units in β/α type proteins with 75% accuracy. The location of super-secondary structure was then used to refine the original (Garnier et al., 1978) secondary structure prediction resulting in an 8.8% improvement, which correctly assigned 83% of secondary structure elements in 14 proteins. Slight modifications to the Garnier et al. method arc suggested, producing a more accurate identification of protein class and a better prediction for β/α. type proteins. A method for the incorporation of hydrophobic information into the prediction is also described.     

High-cell-density fermentation studies of a recombinantE. coli that expresses atrial natriuretic factor

Summary Studies are presented on the fermentation of recombinantEscherichia coli that express rat atrial natriuretic factor (ANF) as a fusion protein. Our objective was to achieve high cell density while maintaining ANF expression at the same level as observed in shake flasks. Improved fermentation conditions included: maintaining glucose concentrations at 1 g/l, using an enriched medium, adding concentrates of medium throughout the fermentation, and blending oxygen for adequate aeration. Cell densities of 12 g/l (dry weight) were achieved, which represented a 10-fold increase over non-improved conditions, while maintaining ANF levels at 7 mg/g of dry cell mass. When galactose was used as an initial carbon source or as a feed supplement, there was a 2-3-fold increase in the expression of ANF from these high-cell-density fermentations. The recombinant ANF was biologically active.     

Amino acid sequence of rabbit ventricular myosin light chain-2: Identity with the slow skeletal muscle isoform

Many studies have established a correlation of differences in the activities of various muscle types with differences in the expression of myosin isoforms. In this paper we report the sequence determination of myosin light chain-2 from rabbit slow skeletal (LC2s) and ventricular (LC2v) nmscles. We sequenced tryptic peptides from LC2v which account for all except a few terminal amino acid residues. The major part (87 residues) of the rabbit LC2s sequence, obtained from tryptic and cyanogen bromide (CNBr) peptides, was found to be identical to rabbit LC2v. Our results provide the first sequence information on LC2s from any species, and lend strong support to the hypothesis that LC2s and LC2v are identical. Comparisons of rabbit LC2v and LC2s with rabbit LC2f (from fast skeletal muscle), and also with chicken LC2f and LC2v, show clearly that LC2s and LC2v from mammalian and avian species are more closely related to each other than they are to LC2f isoforms from the same species.