Comparison of proteins from Mycobacterium fortuitum, Mycobacterium nonchromogenicum and Mycobacterium terrae using flat bed electrophoresis.

Polyacrylamide gel electrophoresis of bacterial lysates in a flat bed gives a linear relationship between 1n mol. wt of the proteins and the square root of their migration distances, thereby allowing standardization of different electrophoresis runs and precise comparison between homologous bands. The results obtained with Mycobacterium fortuitum, M. terrae and M. nonchromogenicum strains were used in numerical analysis. Mycobacterium fortuitum and M. nonchromogenicum showed a greater internal similarity than M. terrae, while two strains of the latter clustered with M. nonchromogenicum. The method described allows the comparison of mycobacteria with different generation times and provides a large number of good characters for numerical taxonomy.     

Isolation and partial characterization of heparan sulphate proteoglycan from the human glomerular basement membrane.

Heparan sulphate proteoglycan was solubilized from human glomerular basement membranes by guanidine extraction and purified by ion-exchange chromatography and gel filtration. The yield of proteoglycan was approx. 2 mg/g of basement membrane. The glycoconjugate had an apparent molecular mass of 200-400 kDa and consisted of about 75% protein and 25% heparan sulphate. The amino acid composition was characterized by a high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulphonic acid yielded core proteins of 160 and 110 kDa (and minor bands of 90 and 60 kDa). Alkaline NaBH4 treatment of the proteoglycan released heparan sulphate chains with an average molecular mass of 18 kDa. HNO2 oxidation of these chains yielded oligosaccharides of about 5 kDa, whereas heparitinase digestion resulted in a more complete degradation. The data suggest a clustering of N-sulphate groups in the peripheral regions of the glycosaminoglycan chains. A polyclonal antiserum raised against the intact proteoglycan showed reactivity against the core protein. It stained all basement membranes in an intense linear fashion in immunohistochemical studies on frozen kidney sections from man and various mammalian species.     

The structure of V alpha and J alpha segments in the mouse.

Antigen receptors on most T-cells are heterodimeric glycoproteins, comprised of an alpha chain and a beta chain. These chains are encoded by discontiguous variable (V), diversity (D) and joining (J) gene segments that rearrange to produce a contiguous and functional alpha or beta chain gene. To investigate the size and diversity of the germline repertoire of alpha-chain gene segments, we have characterized and sequenced 20 alpha chain cDNAs. Among these cDNA clones, we have found 4 J alpha and 4 V alpha sequences that have not yet been described. The relationship of these "new" gene segments to those already characterized is discussed.     

Diversity of the mouse T cell receptor Cγ1 gene: structural analysis in C57BL/Ka

We have isolated an unusual T cell receptor chain cDNA clone (7.1) from a library made from RNA derived from adult thymus of C57BL/Ka mice. This cDNA clone corresponds to the appropriately processed C1 constant region exons preceded by 1.5 kb of J-C1 intron. The 7.1 coding region is extremely homologous to the C1 gene of BALB/c mice, differing at the protein level by a single deletion (alanine 139) and a single substitution. This latter change eliminates the sole N-linked sugar attachment site, providing a basis for strain-specific glycosylation patterns. The J-C1 intronic region contains two DNA segments (termed J1 and J2) that are highly reminiscent of joining (J) segments; both have potentially functional recombination and donor splice sequences flanking an open reading frame. Northern analysis suggests that 7.1 may be derived from a large, variable region-containing precursor.     

Lycorine: a eukaryotic termination inhibitor?

The effect of the alkaloid lycorine on viral protein synthesis was studied in poliovirus-infected HeLa cells. The incorporation of [3H]leucine was inhibited by lycorine in a dose-dependent way, although lycorine never completely abolished translation. Using polyacrylamide gel electrophoresis, the viral proteins were identified as derived from the P1 (5' terminal), P2 (middle), or P3 (3' terminal) region of the poliovirus translation unit. The residual labeling of viral proteins in the presence of lycorine was mainly due to synthesis of P1 proteins and slightly less to P2 proteins, while virtually no P3-derived proteins were made. It is suggested that lycorine may act at the level of termination.     

Avidin-HRP conjugates in biotin-avidin immunoenzyme cytochemistry

Avidin-HRP conjugates were prepared, analysed and tested for avidin-biotin immunocytochemistry. Suitable biotinylation of enzymes, antigens and antibody was obtained by reacting biotin at equimolar ratio to epsilon aminogroups in proteins. The avidin-biotin interaction was used for immunocytochemical detection of phenomena in the field of immunology, i.e. immune complex trapping, specific antibody forming cells and in serology for the cytochemical detection of human auto-antibodies to basement membrane components. Avidin-HRP conjugation using the two step glutaraldehyde method gave a very small amount of monomeric, low molecular weight conjugate with excellent performance. Avidin-HRP conjugation using the periodate method was modified at two points. The first modification concerns the molar ratio of avidin to HRP in the reaction mixture which was brought to about equimolarity. The second modification concerns the periodate concentration which was decreased five fold, ten fold and twenty fold. Decreasing the periodate concentration decreased the amount of polymeric conjugate. Optimal amounts of monomeric, low molecular weight conjugate were obtained with a ten fold decrease of the periodate concentration. Comparable cytochemical results were obtained with monomeric conjugates obtained using both preparation methods.     

Changes in ascorbic acid content and ascorbate peroxidase activity during the development of Acetabularia mediterranea

We cloned ras-related sequences from goldfish genomic libraries constructed as recombinants using the lambda phage. Restriction enzyme mapping of the clones obtained revealed three kinds of ras-related sequences among approximately 350,000 genomic clones. One of these clones was partially sequenced. Comparison with the nucleotide sequences of mammalian ras genes showed that the determined sequences covered the predicted amino acid coding regions and parts of the intervening regions. The predicted amino acid sequences of the cloned ras-related goldfish gene suggested that the coding region is localized separately in DNA, and that its exon-intron boundaries are exactly the same as those of corresponding mammalian genes. The nucleotide and amino acid sequences of the goldfish ras-related gene may have extensive homologies to mammalian p 21 protein. Among the three mammalian ras proteins, the predicted amino acid sequence of the sequenced ras-related goldfish clone is most closely homologous (96%) to the Kirsten ras protein. Differences in the predicted amino acid sequence were greatest in the sequence predicted from the fourth exon; fewer differences were found in the sequence from the third exon, and only slight or no differences were found in the sequence predicted for the first and second exons. The 12th and 61st amino acids from the N-terminal of the protein, which are thought to be critical positions for GTP binding and catalysis, are both conserved in the goldfish protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Avidin-HRP conjugates in biotin-avidin immunoenzyme cytochemistry

Summary Avidin-HRP conjugates were prepared, analysed and tested for avidin-biotin immunocytochemistry. Suitable biotinylation of enzymes, antigens and antibody was obtained by reacting biotin at equimolar ratio to epsilon aminogroups in proteins. The avidin-biotin interaction was used for immunocytochemical detection of phenomena in the field of immunology, i.e. immune complex trapping, specific antibody forming cells and in serology for the cytochemical detection of human auto-antibodies to basement membrane components. Avidin-HRP conjugation using the two step glutaraldehyde method gave a very small amount of monomeric, low molecular weight conjugate with excellent performance. Avidin-HRP conjugation using the periodate method was modified at two points. The first modification concerns the molar ratio of avidin to HRP in the reaction mixture which was brought to about equimolarity. The second modification concerns the periodate concentration which was decreased five fold, ten fold and twenty fold. Decreasing the periodate concentration decreased the amount of polymeric conjugate. Optimal amounts of monomeric, low molecular weight conjugate were obtained with a ten fold decrease of the periodate concentration. Comparable cytochemical results were obtained with monomeric conjugates obtained using both preparation methods.In honour of Professor P. van Duijn     

Mechanisms of cell differentiation: Affinity of a morphogenetic factor to DNA

Summary A morphogenetic factor which induces inTriturus gastrula ectoderm tissues which are derived from mesoderm and endoderm has been extracted from chicken and amphibian embryos. The factor which is protein in nature has been obtained from chicken embryos in a highly purified state.The biological activity of the chicken factor is partially inhibited when the factor is combined with chicken DNA or sonicated chicken DNA.When the 3H-labelled factor is combined with sonicated DNA and then centrifuged on a sucrose gradient the factor migrates in part with the DNA. This indicates that the factor is bound to DNA.The inferences from these results are discussed with regard to the possible mechanism of action of the factor and the molecular mechanism of differentiation.