Effects of co-expressing chaperone BiP on functional antibody production in the baculovirus system.

The assembly pathway of the insect cell Spodoptera frugiperda (Sf-9) was engineered to include expression of the murine chaperone immunoglobulin heavy chain binding protein (BiP) using the baculovirus vector. The impact of BiP coexpression on the production and secretion of functional and soluble recombinant immunoglobulin IgG levels was evaluated. Recombinant BiP was found to associate specifically with immunoglobulins in immunoprecipitation studies. Coinfection of insect cells with a BiP-containing baculovirus and baculoviruses coding for two different murine IgG proteins increased intracellular functional antibody activity levels substantially above the levels observed in the absence of BiP. Soluble intracellular immunoglobulin levels were found to increase as well. However, secreted functional antibody levels did not increase significantly. Also, degradation of heavy chain immunoglobulin in insect cells was indicated by the accumulation of lower molecular weight immunoglobulins at 4 days postinfection. Coexpression of light chains reduced the level of these lower molecular weight immunoglobulins while BiP coexpression led to enhanced levels. These findings suggest that coexpressed BiP can increase intracellular soluble and functional antibody yields but that secretion in the baculovirus-insect cell system must be limited at some post-translational step.

Male-specific expression of mouse sex-limited protein requires growth hormone, not testosterone.

Sex-limited protein (Slp), an isoform of mouse complement component C4, is expressed predominantly in liver and nearly exclusively in sexually mature males or testosterone-treated females. It is encoded by a gene (C4-Slp) whose hormonal dependence has been attributed to an androgen-responsive transcriptional enhancer introduced accidentally, alongside the C4-Slp promoter, in the guise of the 5' long terminal repeat of an ancient retrovirus. We demonstrate that the pronounced rise of C4-Slp mRNA promoted by androgens in the liver is due to nuclear factors acting at a transcriptional stage. Curiously, hypophysectomized animals of either sex fail to express the gene and are refractory to testosterone. However, gene expression at male levels is restored even more promptly by injections of growth hormone alone. Additionally, animals carrying an ubiquitously expressed human growth hormone transgene lack C4-Slp mRNA and are insensitive to testosterone treatment. That growth hormone is sufficient to induce expression in a manner independent of androgen-receptor activity is shown by the hormonal treatment of Tfm mice. These androgen receptor-defective animals lack C4-Slp mRNA, which however can be fully induced by growth hormone injections. We conclude that the sexual dimorphism of C4-Slp expression employs liver nuclear mediators distinct from those directly instructed by androgens and is brought about by the intermittent rise of growth hormone, dictated by testosterone.

The mouse mammary tumour virus long terminal repeat encodes a type II transmembrane glycoprotein.

Superantigens are products of bacterial or viral origin which stimulate large numbers of T cells as a consequence of the interaction of particular V beta chains of the T cell receptor with class II major histocompatibility complex (MHC) molecules and superantigen on the stimulating cell. The Minor lymphocyte stimulatory (Mls) antigens, originally discovered as strong lymphocyte stimulatory determinants in vitro and subsequently shown to delete T cells expressing specific V beta chains during development, have recently been shown to be genetically linked to endogenous mouse mammary tumour viruses (MTVs). This stimulation is effectuated by an unidentified product encoded by an open reading frame (orf) present in the 3' long terminal repeat (LTR) of MTVs. Using in vitro translation in the presence of rough microsomal vesicles, we show that (i) the orf of MTV encodes a type II transmembrane glycoprotein (N-terminus intracellular, C-terminus extracytoplasmic), and (ii) a cotranslationally secreted orf protein is not produced. We have also isolated and sequenced several endogenous MTV orfs (MTV-1, MTV-6 and MTV-13) which are involved in the deletion of V beta-bearing T cells; each of these sequences are nearly identical to each other. These observations, together with sequence comparisons of several orf genes, lead to a model of action of viral superantigens.

A single base deletion in the Tfm androgen receptor gene creates a short-lived messenger RNA that directs internal translation initiation.

Testosterone-resistant male mice hemizygous for the X-chromosome-linked mutant gene Tfm express detectable but severely reduced levels of androgen receptor mRNA, amounting to about 10% of the level found in normal male littermates. No structural abnormality could be identified in the coding region of the messenger by a series of RNase-protection assays. However, cell-free translation of RNAs transcribed in vitro from enzymatically amplified overlapping segments of exon 1 revealed a truncated receptor protein and helped to localize the site of premature termination. Sequence analysis of the relevant DNA segment disclosed that deletion of a single nucleotide in the hexacytidine stretch at position 1107-1112 alters the reading frame of the messenger and introduces 41 missense amino acids before a premature termination codon at position 1235-1237. Separately initiated carboxyl-terminal polypeptides are synthesized in vitro, starting probably at the in-frame AUG codon 1507-1509, which lies in a favorable context for translation initiation, and at the non-AUG codon 1144-1146. Transcriptional impairments of the Tfm gene were ruled out by a quantitative analysis of enzymatically amplified nuclear RNA precursors. No other change could be identified by sequencing the complete coding region of Tfm cDNA. The finding of the unsuspected termination codon and the evidence of internally initiated carboxyl-terminal polypeptides reconcile previous conclusions and account for all known phenotypic properties of the mutation.

[Structure of messenger RNA of androgen receptor in mice and molecular characterization in Tfm mutant]. / Structure de l'ARN messager du récepteur des androgènes de Souris et caractérisation moléculaire du mutant Tfm.

Complementary DNA clones covering the complete coding region of the mouse androgen receptor were assembled by enzymatic amplification (PCR) from testicular RNA and genomic DNA and fully sequenced. The deduced amino acid sequence departs from the rat sequence at 21 positions, 20 of which are in the amino-terminal trans-activation domain. A single 10 kb long messenger RNA containing a 3' noncoding portion longer than 5 kb was detected. The murine cDNA sequence provided the basis to examine the testicular feminization (Tfm) mutation at the molecular level. The androgen receptor messenger RNA level was found reduced about 10-fold in the Tfm mice. The expression of the mutant receptor is affected at a post-transcriptional level. A single base deletion in the hexanucleotide stretch 1107-1112, not far from the 3' end of exon 1, introduces a frame-shift that leads to premature termination of the AR protein. Separately initiated polypeptides containing the DNA-binding and the steroid-binding domains of the androgen receptor are produced in vitro by using strong internal translation initiation sites. These carboxyl-terminal polypeptides have the characteristics of the shortened form of the receptor previously described in the tissues of Tfm mice.

Molecular cloning of human C1 inhibitor: sequence homologies with alpha 1-antitrypsin and other members of the serpins superfamily.

Genetic and acquired diseases in man show that the proteolytic activity of the complement component C1 is crucially regulated by C1 inhibitor (C1-INH), a plasma protein whose suspected relatedness to other serine proteinase inhibitors (serpins) contrasts with its atypically large size and high degree of glycosylation. Indeed we have found that the C1-INH polypeptide precursor synthesized in a cell-free system is a 64-kDa protein, hence it exceeds the length of the precursor forms of typical serpins. Seeking more conclusive sequence information and a probe for the structural locus, we isolated C1-INH cDNA clones from a library representing size-enriched human liver mRNA. Nucleotide sequence analysis of a clone covering the carboxyterminal half of C1-INH conclusively documents the relatedness of this protein with the serpins, and reveals 27% amino acid identity with alpha 1-antitrypsin.

Leukocyte recovery with short-chain RNA fragments in cyclophosphamide-treated rabbits.

Single-stranded short-chain RNA fragments, obtained by mild degradation of purified Escherichia coli ribosomal RNA(s) with pancreatic RNase A, exhibit particular biologic activities in vitro and in vivo. In vitro, these RNA fragments are used by DNA-dependent DNA polymerase I as primers to initiate the replication of DNA(s) isolated from rabbit bone marrow and spleen; they are inactive with DNA isolated from several normal tissues and cancerous cells. Administered iv, RNA fragments restore a normal level of circulating leukocytes in rabbits with high doses of cyclophosphamide (CP). Granulocyte/lymphocyte balance, upset by daily CP administration, is also restored during the increase of both types of cells. No toxicity is observed, and numerous repeated doses of RNA fragments show no cumulative effect and do not lead to loss of leukopoietic stimulating activity. Tumor-bearing mice can be protected by RNA fragments against the toxic effect of CP without impeding the anticancer activity of this drug.

Correlation between in vitro DNA synthesis, DNA strand separation and in vivo multiplication of cancer cells.

The chemicals 9, 10-dimethylbenzanthracene (DMBA), ethionine, daunorubicin, actinomycin D, 1-(2-chloroethyl-1)-nitrosourea (CCNU), steroids, croton oil and dimethyl-sulfoxide (DMSO) were used in order to correlate their effect on the in vitro synthesis of normal and cancer DNA, on DNA strand separation and on accelerated in vivo multiplication of cancer cells. All of the compounds tested strongly stimulate the synthesis of cancer DNA in vitro catalyzed by DNA-dependent DNA polymerase I and measured as an acid-precipitable labeled product. Under the same conditions, the synthesis of DNA originating from healthy tissues is only slightly enhanced, except in the case of croton oil and DMSO. These substances are almost equally active on cancer and normal DNA. Although both cancer and normal DNA contain a large amount of double-stranded regions, the extent of DNA strand separation measured by the increase in UV absorbance (hyperchromicity) in the presence of each compound tested is much higher for all cancer DNA than for corresponding normal DNA. In contrast, DMSO and croton oil do not appear to distinguish cancer DNA from normal DNA. Additive and differential effects of various compounds on cancer DNA strand separation can be observed. Small doses of DMBA and CCNU stimulate the multiplication of Ehrlich ascites tumor cells in vivo in mice. There is thus a possible correlation between DNA strand separation, DNA synthesis, multiplication and differentiation of cancer cells in the presence of the above compounds, which is different from the response of normal cells to these compounds.

[Degradation of ribosomal RNA from Escherichia coli by ribonuclease U2 and in vitro transcription of "RNA-fragments" into complementary DNA]. / Découpage des ARN ribosomiques d'Escherichia coli par la ribonucléase U2 et transcription in vitro des "ARN-fragments" en ADN complémentaire.

Under well defined conditions ribosomal RNAs purified from Escherichia coli can be degraded by ribonuclease U2 giving rise to RNA fragments of 60--70 nucleotides. In vitro, these fragments are efficiently transcribed into a complementary DNA by DNA polymerase RNA dependent, partially purified from extracts of E. coli. In vivo, "RNA-fragments-U2" inhibit the development of plant tumors.

[RNA-fragments, necessary primers for the in vitro replication of DNA]. / ARN-fragments, amorceurs nécessaires à la réplication in vitro des ADN

Partial degradation of ribosomal RNAs (E. coli rich in purine nucleotides) by different ribonucleases gives rise to the appearance of several families of RNA-fragments which after separation on "Sephadex G 25" were analyzed for base ratio, size and biological activity. In the presence of DNA dependent DNA polymerase, RNA-fragments act as primers for in vitro replication of DNAs from numerous sources.

Isolation of the tumor-inducing RNA from oncogenic and nononcogenic Agrobacterium tumefaciens.

Two RNA fractions have been isolated and purified from both oncogenic and nononcogenic strains of Agrobacterium tumefaciens. Both RNAs are capable of inducing the formation of transplantable tumors when introduced at wound sites in stems of Datura stramonium plants. One of these RNA fractions was found to be bound to an RNA-directed DNA polymerase, while the other was associated with the bacterial DNA. Physical evidence suggests that both are single stranded and small in size; linear sucrose gradients show that their size corresponds to a value of 5-6 S. A concentration of 4-5 mug of the RNAs dissolved in 0.01 ml of water is effective in initiating the formation of transplantable tumors in Datura plants.

Transformation of Agrobacterium tumefaciens into a non-oncogenic species by an Escherichia coli RNA.

Transforming RNA excreted by showdomycin-resistant Escherichia coli induces a persistent, heritable, and spectacular change in Agrobacterium tumefaciens B(6), a bacterium that carries the oncogenic principle for tumor induction in plants. Transformants possessing new physiological and biochemical properties have completely or partially lost the capacity for tumor induction. They synthetize new ribosomes whose components are profoundly modified. On the basis of biological and biochemical characteristics, one is inclined to consider the completely transformed Agrobacterium tumefaciens as a "new species".

Drastic alteration of ribosomal RNA and ribosomal proteins in showdomycin-resistant Escherichia coli.

In a mutant of Escherichia coli resistant to showdomycin, both the 50S and 30S ribosomal subunits contain RNA species in which the purine concentration greatly exceeds that of pyrimidines. The same is true for total rapidly-labeled RNA. The modified ribosomal RNA hybridizes poorly with homologous DNA, which is apparently unchanged in base composition. Acrylamide gel electrophoresis of mutant ribosomal proteins shows a highly altered protein pattern for both ribosomal subunits, although the activity of these ribosomes is not decreased.