The biogenesis and assembly of bacterial membrane proteins

Bacterial proteins in the inner and outer membranes differ dramatically in their architecture. Although both types of proteins are transported across the inner membrane through a common pore, recent studies have identified distinct factors that target them to transport sites and catalyze proper folding.     

Purification of the 56-kDa component of the bacteriophage T7 primase/helicase and characterization of its nucleoside 5'-triphosphatase activity

Bacteriophage T7 gene 4 protein, purified from phage-infected cells, consists of a mixture of a 56- and a 63-kDa species that provides primase and helicase activities for T7 DNA replication. The 56-kDa species has been purified 1800-fold from Escherichia coli cells containing a plasmid that encodes this gene 4 protein. The purified 56-kDa protein is homogeneous, as determined by denaturing gel electrophoresis, and is monomeric in its native form, as indicated by gel filtration. The binding of the 56-kDa protein to single-stranded DNA is stimulated by nucleoside 5'-triphosphates, as is the case for a mixture of the two molecular weight species. In the presence of DNA, the 56-kDa protein preferentially hydrolyzes dTTP (Bernstein, J. A., and Richardson, C. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 396-400). Since nucleoside 5'-triphosphatase activity is necessary for both helicase activity and for translocation of gene 4 protein to primase recognition sites, we have characterized this activity using the 56-kDa protein alone. In the DNA-dependent hydrolysis reaction, the enzyme displays a Km of 10 mM for dTTP, and a Vmax of 2.9 x 10(-5) M/min/mg of protein (at 2.5 micrograms/ml). There is little cooperativity with respect to dTTP binding (Hill coefficient = 1.1) except in the presence of ribonucleoside 5'-triphosphate, an inhibitor of dTTP hydrolysis (Hill coefficient greater than 1.5). The apparent KD for single-stranded circular DNA is 0.2 microM. The active species in dTTP hydrolysis is an oligomer of at least two subunits, as indicated by the effect of enzyme concentration upon the rate of DNA-dependent hydrolysis. The 56-kDa protein also catalyzes DNA-independent hydrolysis of dTTP with a Km of 0.11 mM and a Vmax of 1.3 x 10(-7) M/min/mg of protein (at 8 micrograms/ml). The active species in DNA-independent dTTP hydrolysis is also an oligomer.     

Non-random in vitro 7;14 translocations detected in a routine cytogenetic series. 12 Examples and their possible significance

Summary This study describes 12 examples of translocations between chromosomes 7 and 14 in short-term peripheral blood lymphocyte cultures from 10 patients investigated in a routine cytogenetic series. Only one constant breakpoint was found on 14q, and chromosome 7 had two constant breakpoints, one on 7p and the other on 7q. The cause and true significance of such nonrandom in vitro chromosome translocations is not known at present, but one may speculate as to their possible indication of heterozygosity for a chromosome instability syndrome and thus a predilection for the development of lymphoid or other malignancy.     

Growth and differentiation of primary rat kerationocytes on synthetic membranes

Summary The attachment, proliferation, and differentiation of primary cultures of kerationcytes isolated from murine epidermis were monitored after purified cell suspensions were seeded and incubated in vitro on various synthetic membranes. Concomitant studies of the effects of attachment factors added to synthetic membranes before use as substrata for keratinocytes were also done. The study demonstrated that a synthetic membrane composed of nylon was superior to other membranes and to plastic control culture vessels in supporting the growth of murine keratinocytes. Although laminin enhanced initial attachment and proliferation of cells on nylon membranes, the untreated substratum was more effective for extended incubation. Stratification and differentiation of these kerationcytes on the nylon substratum was enhanced by raising confluent cultures (7 d) to the air-medium interface so that they were in contact with medium only from the bottom. Cultures raised for 14 d produced many morphologic markers of the epidermis and closely resembled the architecture of this tissue in situ. This investigation was supported in part by the Johns Hopkins Center for Alternatives to Animal Testing and by the United States Army Medical Research and Development command, contract DAMD17-82-C-2198. The views, opinions or findings contained in this report are those of the authors and should not be construed as position, policy or decision of the agency supporting the research.     

Transfer of human and murine globin-gene sequences into transgenic mice.

We have studied the transfer of human and murine globin gene sequences into fertilized mouse oocytes by microinjection. Germline transmission was demonstrated for the human delta- and beta-globin genes contained in the bacteriophage lambda H beta G1. Expression of these human globin-gene sequences was not detectable in either erythroid or nonerythroid tissues. A recombinant plasmid containing the murine beta maj promoter region coupled to the prokaryotic coding sequence for galactokinase was also successfully transferred to two mice, and stable germline transmission of integrated DNA was demonstrated for at least 3 generations. Despite the presence of a murine globin-promoter sequence, expression of the mouse beta maj galactokinase fusion gene was not observed in primary or secondary animals in erythroid or nonerythroid tissues. Analysis of primary and secondary animals from both series of injections revealed extensive de novo methylation in the integrated microinjected DNA. Administration of 5-azacytidine to mice containing the mouse beta maj-promoted galactokinase gene resulted in partial hypomethylation was associated with an apparent two- to threefold increase in galactokinase (gal K) gene expression.     

The preparation of estradiol-17 beta sulfates with triethylamine-sulfur trioxide

Reaction of estradiol-17 beta with triethylamine-sulfur trioxide in pyridine gives exclusively monosulfation at the C17-hydroxyl group with the preparation of 17 beta-sulfooxyestra-1,3,5(10)-trien-3-ol triethylammonium salt (V). The structural assignment suggested by spectroscopic measurements was confirmed by synthetic studies. (Formula: see text) A synthesis of 3-sulfooxyestra-1,3,5(10)-trien-17 beta-ol triethylammonium salt (II) has been accomplished based on the preparation of 17 beta-formyloxyestra-1,3,5(10)-trien-3-ol (XIII). Fusion of the 3-sulfate triethylammonium salt II gives rise to the 17-sulfate triethylamine salt V. The preparation of estradiol-17 beta disulfate has also been achieved.     

Poliovirus mutant that does not selectively inhibit host cell protein synthesis.

A poliovirus type I (Mahoney strain) mutant was obtained by inserting three base pairs into an infectious cDNA clone. The extra amino acid encoded by the insertion was in the amino-terminal (protein 8) portion of the P2 segment of the polyprotein. The mutant virus makes small plaques on HeLa and monkey kidney (CV-1) cells at all temperatures. It lost the ability to mediate the selective inhibition of host cell translation which ordinarily occurs in the first few hours after infection. As an apparent consequence, the mutant synthesizes far less protein than does wild-type virus. In mutant-infected CV-1 cells enough protein was produced to permit a normal course of RNA replication, but the yield of progeny virus was very low. In mutant-infected HeLa cells there was a premature cessation of both cellular and viral protein synthesis followed by a premature halt of viral RNA synthesis. This nonspecific translational inhibition was distinguishable from wild-type-mediated inhibition and did not appear to be part of an interferon or heat shock response. Because the mutant is recessive, our results imply that (at least in HeLa cells) wild-type poliovirus not only actively inhibits translation of cellular mRNAs, but also avoids early inhibition of its own protein synthesis. Cleavage of the cap-binding complex protein P220, which has been associated with the selective inhibition of capped mRNA translation, did not occur in mutant-infected cells. This result supports the hypothesis that cleavage of P220 plays an important role in normal poliovirus-mediated translational inhibition.     

Characterization of a retrovirus shuttle vector capable of either proviral integration or extrachromosomal replication in mouse cells.

A retrovirus shuttle vector is described that contains the dominant selectable neo gene which confers resistance to kanamycin in bacteria and to the drug G418 in animal cells. The bacterial supF gene and the origins of DNA replication from polyomavirus and the ColE1 replicon also have been included in this vector. Infection of normal rodent cells results in single-copy proviral integration, whereas infection of mouse (MOP) cells expressing polyoma large T antigen results in extrachromosomal replication of the DNA form of the virus. The copy number of the extrachromosomal circles in MOP cells varies from 0 to 100 copies per cell. G418-resistant MOP cells lose their drug-resistant phenotype after passage under nonselective conditions, suggesting that maintenance of the extrachromosomal circles is unstable. The extrachromosomal form of the virus can be recovered as plasmids in Escherichia coli. Two-thirds of the circles analyzed were found to be structurally intact. The others have undergone rearrangements including deletions and insertions. The bacterial supF gene was found to be intact in the majority of recovered plasmids. The data presented here suggest that these retroviruses should be useful as gene transfer vectors for animal cells in culture or in vivo.     

Germline VH genes in an a3 rabbit not typical of any one VHa allotype

We have undertaken investigations in the rabbit of VH genes that may be responsible for the observations of VHa allotypes unexpected from an animal's pedigree (latent allotypes). A short cDNA probe was prepared and shown to be specific for VHa2 mRNA. Southern analyses with short and large probes were unrevealing but screening of a lambda phage library from a VHa3-expressing animal identified a number of unusual genes. These VH genes are remarkable in that they are far closer to one another in the genome (in one case 3085 bps) than VH genes reported in mouse or man, they are highly homologous over long stretches of sequence, and they encode proteins not typical of any one VHa allotype. Proteins similar to the sort encoded by these genomic V-regions may explain some of the observations of latent allotype. Our data suggest that the allelic behavior of VHa allotypes is not due to allelism of a regulatory mechanism that acts upon identical VH genes in rabbits of different VHa phenotypes.     

Influence of antibody isotype on passive serotherapy of lymphoma

We assessed the in vivo anti-tumor effectiveness of monoclonal antibodies of different isotypes. Starting with a hybridoma cell secreting an IgG3 anti-Thy-1.1 antibody, we isolated three variant hybridoma cell lines secreting anti-Thy-1.1 antibody of the IgG1, IgG2a, and IgG2b isotypes. Each antibody displayed identical antigen binding properties, but differed in their ability to mediate in vitro lysis of Thy-1.1+ AKR/J SL2 lymphoma cells. In assays of complement dependent cytotoxicity, the relative activity of each antibody isotype was IgG2a = IgG2b greater than IgG3 greater than IgG1. In assays of antibody-dependent cell-mediated cytotoxicity when using non-immune spleen cells as effectors, the relative activities were IgG2a greater than or equal to IgG2b greater than IgG1 greater than IgG3. Infusion of equivalent amounts of each antibody (1.5 mg) in AKR/Cum (Thy-1.2+) mice inoculated subcutaneously with 3 X 10(5) AKR/J SL2 lymphoma cells resulted in significant inhibition of tumor growth only in mice treated with IgG2a antibody. However, the antibodies were cleared at different rates, with the IgG2a antibody having the slowest clearance. When antibody doses were adjusted to achieve equivalent serum levels 24 hr after infusion, all of the antibody isotypes exhibited at least some anti-tumor activity, although IgG2a antibody was again the most effective. These studies demonstrate that the difference in anti-tumor activity between antibodies of different isotypes may result from differences both in their serum clearance rate and their ability to interact with host effector mechanisms.