Human retroviral sequences on the Y chromosome.

Novel endogenous human retroviral sequences were cloned by low-stringency hybridization, using the pol gene of endogenous human retrovirus 51-1. One clone, lambda NP-2, contained gag, pol, env, and long terminal repeat sequences related to the corresponding portions of clone 51-1 and the closely related full-length endogenous human retrovirus 4-1. The sequence of the env gene of NP-2 was 73% homologous to that of 4-1. Genomic Southern blots of male and female DNAs showed that NP-2 is located on the Y chromosome and that the Y chromosome also contains one other sequence closely related to the env and 3' flanking regions of NP-2. Conservation of flanking DNA suggests that the second Y chromosome copy of the NP-2 env sequence arose by gene duplication rather than provirus insertion.     

Depressed natural cytotoxicity but normal natural killer cytotoxic factor (NKCF) production by mononuclear cells derived from patients with hepatocellular carcinoma

Summary This study investigated the relation between the production of natural killer cytotoxic factors (NKCF) and the phenomenon of natural killing (NK) activity against target K562 cells. Two different models of defective NK cell activity were employed. In the first instance, cytotoxic activity of mononuclear cells (MN) derived from patients with hepatocellular carcinoma was compared to the ability of these cells to produce NKCF. Although direct cytotoxicity was considerably impaired in these patients, the ability of their MN to produce NKCF when stimulated with K562 cells was found to be normal. In the second model, MN treated with the lysosomotropic drug monensin showed considerably reduced direct cytotoxic activity, although they were capable of producing normal amounts of NKCF when activated by K562 cells. These results therefore indicate that there is no correlation between NK activity and corresponding NKCF release, and suggest that NKCF production and activity is independent of direct NK cytotoxic activity.     

Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha

We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.     

Synthesis of functional human immunodeficiency virus tat protein in baculovirus as determined by a cell-cell fusion assay.

The human immunodeficiency virus tat protein is a strong trans-activator of the expression of mRNAs originating from the viral long terminal repeat. We have expressed the first 72 amino acids (coding exon 1) of this protein in eucaryotic Spodoptera frugiperda SF9 cells by using a baculovirus vector, Autographa californica nuclear polyhedrosis virus. We show that the baculovirus vector stably produced the 72-amino-acid form of the tat protein but was unable to stably synthesize a larger 101-amino-acid full-length version of the same polypeptide. The 72-amino-acid tat protein, when introduced into mammalian fibroblasts by using a cell-cell fusion technique, functionally trans-activated the expression of the human immunodeficiency virus long terminal repeat.     

The in vitro effects of histamine and metiamide on neutrophil motility and their relationship to intracellular cyclic nucleotide levels.

Histamine at concentrations of 1 x 10(-5) M to 5 x 10(-5) M consistently increased neutrophil movement as measured in Boyden chambers. This effect was entirely caused by stimulation of chemokinesis (stimulated random migration) and true chemotaxis was inhibited by these concentrations. This inhibition of chemotaxis could be abolished by pretreatment with metiamide, an H-2 receptor antagonist, and levamisole, but not by diphenylhydramine, an H-1 receptor antagonist. Metiamide at similar concentrations produced a mild stimulation of chemokinesis but has no effect on true chemotaxis. The histamine effects on neutrophil motility were associated with increased levels of intracellular cAMP wehreas cAMP levels were unaffected. Agents known to elevate intracellular cAMP levels produced effects on neutrophil motility similar to those of histamine. It is suggested that histamine exerts a 2-fold effect on neutrophil motility mediated via an H-2 receptor site and associated with elevated levels of cAMP.     

Inherited deficiency of the sixth component of complement: a silent or null gene

Four families have been studied, some members of which have inherited deficiency of the sixth component of complement. The genetically determined electrophoretic variants of C6 were evaluated in all family members. Seven individuals were found who did not have the variant found in the serum of the parent from whom they inherited the deficiency. It is inferred that the isolated low levels of C6 in these individuals results from the heterozygous state of a normal C6 variant gene and a silent or null C6 gene; the genes determining electrophoretic variants and the low serum levels of C6 are allelic.     

Sequence-specific toxicity of transfected retroviral DNA.

Experimental gene transfer and viral infections can result in the accumulation of unintegrated DNA in target cells. The effects of such accumulation on target cell metabolism have not been directly studied. The experiments reported in this paper show that transfection of cloned retroviral long-terminal-repeat (LTR) DNA, or of a variety of eukaryotic promoters, into proliferating HeLa cells results in rapid, sequence-specific, and dose-dependent cell death. Plasmids containing the Rous sarcoma virus LTR or the human immunodeficiency virus LTR cloned in pUC-related plasmids are 5 to 10 times more toxic than pUC19. The demonstrated sensitivity of eukaryotic cells to exogenously introduced DNA has important implications for the interpretation of gene transfer experiments and may be relevant to the pathogenic mechanisms in the course of retroviral infections such as AIDS.     

Non-immortalizing P3J-HR-1 Epstein-Barr virus: a deletion mutant of its transforming parent, Jijoye.

The P3J-HR-1 strain of Epstein-Barr virus (EBV) fails to immortalize human lymphocytes. We wished to understand the nature of the genomic alterations which correlated with the loss of this ability. As a first step, the heterogeneity of DNA molecules in the P3J-HR-1 line was eliminated by cell cloning. Then a physical map was prepared of virion DNA from one cell clone, designated FF452-3. By comparison with the genomes of two EBVs, B95-8 and FF41, which are competent to immortalize lymphocytes, we identified a total of eight modifications of BamHI and EcoRI restriction endonuclease fragments of EBV (FF452-3) DNA consisting of insertions, deletions, or loss of a restriction endonuclease recognition site. To determine which of these alterations might be responsible for the loss of transforming phenotype, we examined homologous DNA fragments of the Jijoye strain of EBV, the progenitor of the HR-1 strain which still retains the ability to immortalize lymphocytes. We also studied viral DNA in lymphocytes transformed in vitro by Jijoye virus. Six of the eight alterations were found both in Jijoye and in clonal HR-1 DNA and were presumably genomic traits characteristic of this lineage of EBV. A small deletion in the BamHI-K fragment of HR-1 DNA was not found in Jijoye virion DNA, but this deletion was present in intracellular Jijoye DNA. Thus only one major genomic lesion in HR-1 DNA, a deletion of at least 2.4 x 10(6) molecular weight of DNA from a fused BamHI-H-Y fragment, consistently distinguished Jijoye DNA from its non-immortalizing P3J-HR-1 derivative. This deletion is likely to affect EBV genes which are directly or indirectly involved in immortalizing lymphocytes.