Ribonucleic acid synthesis by Escherichia coli C3000/L after infection by the ribonucleic acid coliphage ZIK/1, and properties of the coliphage-induced double-stranded ribonucleic acid

1. The efficiency of extracting nucleic acids from Escherichia coli after five methods of obtaining cell lysis was determined. 2. The recovery of various nucleic acid species isolated after chromatography on methylated albumin-coated kieselguhr was also examined. 3. Double-stranded coliphage-induced RNA was isolated from infected bacteria and its resistance to ribonuclease digestion under various conditions determined. 4. The involvement of double-stranded RNA during the infection process was demonstrated. 5. The time-course of the syntheses in infected cells of double-stranded RNA, DNA, single-stranded coliphage and 16s ribosomal RNA, transfer RNA and ribosomal 23s RNA was examined. 6. It was demonstrated that the syntheses of DNA, transfer RNA and ribosomal RNA decreased 10-15min. after infection. 7. Synthesis of coliphage RNA commenced 10-15min. after infection and double-stranded RNA was also synthesized from about 10min. after coliphage adsorption.     

Monoclonal antibody against a melanosomal protein in melanotic and amelanotic human melanoma cells

BALB/c mice were immunized with tyrosinase, partially purified in two stages from a human melanoma cell line. A hybridoma was obtained which produced monoclonal antibody (MoAb 1C11) reactive with 8/10 melanoma cell lines and 10/10 primary cultures of human melanocytes, neval cells, and melanomas. Immunoreactivity correlated to a certain extent with tyrosinase activity but not with melanin content. No crossreactivity was obtained with neuroblastoma, medulloblastoma, fibroblasts, keratinocytes, lymphoid cells, or murine melanomas. Purification of the antigen directly from cell lysates with a MoAb 1C11 CNBr-Sepharose affinity column gave a green-brown protein of 56 kDa with no detectable tyrosinase activity. This protein was therefore different from 60 kDa active tyrosinase, identified by enzyme activity and Western blotting with a MoAb derived previously (MoAb 5C12). Unlike 5C12, 1C11 reactivity was not destroyed by pretreatment of the antigen with periodate. Immunogold labelling showed that the 1C11-reactive antigen was associated with melanosomes, and there was close correlation between 5C12 and 1C11 reactivity in resistance to trypsin and in staining various melanocytic cell populations. MoAb 1C11 may therefore recognise a polypeptide epitope in a molecule closely linked to melanin biosynthesis.     

Differential distribution of antigen-specific helper T cells and cytotoxic T cells after antigenic stimulation in vivo. A functional study using limiting dilution analysis

We have developed modified limiting dilution analysis (LDA) techniques that distinguish in vivo Ag-stimulated murine helper T lymphocytes (HTL) and CTL from unstimulated precursor T cells, even those with the same Ag specificity. We refer to these cells that are detectable in the modified LDA as "Ag-conditioned" T cells (cHTL and cCTL). We have used the modified LDA techniques in conjunction with conventional LDA techniques (which enumerate all Ag-specific T cells) to evaluate the in vivo distribution of Ag-conditioned cHTL and cCTL following in vivo sensitization to alloantigens via sponge matrix or skin allografts. In general, we observed the following regarding the distribution of cHTL and cCTL: 1) Ag-conditioned HTL and CTL were detectable only after in vivo sensitization with alloantigen: 2) not all Ag-reactive T cells became conditioned T cells after in vivo Ag deposition; 3) the percentage of Ag-reactive T cells that converted to conditioned T cells after Ag deposition varied among different lymphoid compartments; 4) a high percentage of cHTL, but a low percentage of cCTL, accumulated in regional lymph nodes and spleen; 5) cHTL accumulated in peripheral blood, whereas cCTL did not; 6) Ag-conditioned cHTL were detectable in various lymphoid tissues for greater than 60 days following Ag deposition, whereas cCTL were detectable for only 14 to 20 days; and 7) unlike the other lymphoid sites, the site of Ag deposition accumulated a high percentage of both Ag-stimulated cHTL and cCTL. Furthermore, cHTL and cCTL appeared to reside in phenotypically distinct T cell subsets in that in vivo treatment with anti-L3T4 mAb abrogated the accumulation of HTL, but not CTL, at the site of Ag deposition. These data demonstrate differential compartmentalization of Ag-conditioned cHTL and cCTL subsequent to in vivo Ag deposition. The implications of these findings regarding the monitoring of in vivo immune responses are discussed.     

Apolipoprotein A-I metabolism in subjects with a PstI restriction fragment length polymorphism of the apoA-I gene and familial hypoalphalipoproteinemia

Familial hypoalphalipoproteinemia (hypoalpha), characterized by a decreased high density lipoprotein level, is associated with an increased incidence of premature cardiovascular disease. Restriction fragment length polymorphism analysis of genomic DNA has detected a polymorphism for the PstI restriction endonuclease near the apoA-I gene, with either a 2.2 or a 3.3 kb fragment. The latter has been previously found to occur with significantly higher frequency in probands of families with familial hypoalpha. ApoA-I was isolated from three unrelated subjects with familial hypoalpha and the 3.3 kb PstI polymorphism of the apoA-I gene, and from normal control subjects. The apoA-I from the hypoalpha subjects was structurally normal as determined by amino acid analysis and by two-dimensional gel electrophoresis. When normal apoA-I and hypoalpha apoA-I were simultaneously injected into either normal controls or hypoalpha subjects, both forms of apoA-I were catabolized at the same rate in the same subject, indicating that the hypoalpha apoA-I is also metabolically normal. Analysis of the kinetics of metabolism of apoA-I in the hypoalpha subjects, compared to the normal controls, revealed that the reduced plasma levels of apoA-I were due to an increased apoA-I fractional catabolic rate, and that the synthetic rate was normal. Based on these results, we conclude that the apoA-I gene in these hypoalpha subjects is normal, and the PstI polymorphism near the apoA-I gene, which is associated with familial hypoalpha, is likely to be a marker for a mutant gene closely linked to, but not in, the apoA-I gene.     

Intraspecific evolution of a gene family coding for urinary proteins

Summary The genome of the laboratory mouse contains about 35 major urinary protein (MUP) genes, many of which are clustered on chromosome 4. We have used distance and parsimony methods to estimate phylogenetic relationships between MUP genes from nucleotide sequence and restriction maps. By analyzing coding sequences we show that the genes fall into four main groups of related sequences (groups 1–4). Comparisons of restriction maps and the nucleotide sequences of hypervariable regions that lie 50 nucleotides 5 to the cap sites show that the group 1 genes and probably also the group 2 pseudogenes fall into subgroups. The most parsimonious trees are consistent with the evolution of the array of group 1 and 2 genes by mutation accompanied by a process tending toward homogenization such as unequal crossing-over or gene conversion. The phylogenetic grouping correlates with grouping according to aspects of function. The genomes of the inbred strains BALB/c and C57BL contain different MUP gene arrays that we take to be samples from the wild population of arrays.