High-level expression of the bovine growth hormone gene in heterologous mammalian cells

The gene coding for bovine growth hormone (bGH) was isolated from a lambda-phage library constructed using bovine pituitary DNA partially digested with MboI. Expression of this gene transfected into mouse and monkey cells was studied. CV-1 monkey cells transfected with simian virus 40 (SV40) vectors containing the intact bGH gene, including the putative promoter region, did not express bGH. However, replacement of the bGH promoter with the mouse metallothionein-I (MT) promoter resulted in high-level synthesis and secretion of bGH. These results show that the bGH promoter functions poorly in CV-1 cells but CV-1 cells process and translate the bGH mRNA accurately. The MT-bGH chimeric gene was used to establish permanent bGH-secreting mouse C127 cell lines using the 69% transforming fragment of bovine papilloma virus (BPV) as the vector. One such cell line produced high levels of bGH and secreted it into the medium efficiently. Secreted bGH is processed accurately and is bioactive as judged by its ability to bind to rabbit liver membrane preparations.     

Transient expression of murine interferon-alpha genes in mouse and monkey cells

The coding regions of murine interferon-alpha (IFN-alpha) genes were combined with promoter and 3'-noncoding sequences from other eukaryotic genes. Transient expression of these fusion genes was achieved in monkey COS cells and in a mouse cell line (TOP cells) expressing polyoma virus (Py) large T antigen constitutively. The efficiency of the different expression plasmids was determined by measuring the amount of IFN secreted into the medium. Replacement of the 3'-noncoding region of an IFN-alpha gene by that of the rabbit beta-globin gene resulted in a fourfold higher IFN-alpha production. The SV40 early promoter and the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR) produced similar amounts of IFN-alpha in COS cells. However, a tandem combination of the SV40 enhancer/early promoter and the mouse metallothionein-I promoter appeared fivefold more active than the SV40 early promoter. In TOP cells the MoMLV LTR was found to be threefold more active than the Py early promoter.     

The adeno-associated virus rep gene inhibits replication of an adeno-associated virus/simian virus 40 hybrid genome in cos-7 cells.

A hybrid adeno-associated virus (AAV)/simian virus 40 (SV40) genome is described. In this construct SV40 regulatory sequences, including the early promoter/enhancers and origin of DNA replication, were substituted for the AAV p5 promoter, which normally controls expression of the AAV rep gene. The hybrid genome was phenotypically indistinguishable from wild-type AAV in human cells in the presence or absence of helper virus. Upon transfection into cos-7 cells, which constitutively produced the SV40 tumor antigen, the genome replicated as a plasmid when the SV40 origin was used, although with a low efficiency compared with that of a non-AAV/SV40 replicon. The low level of replication was due to an inhibitory effect of an AAV rep gene product and was specific for replicons containing AAV sequences. Target AAV sequences required for inhibition by rep appeared to reside in the terminal repetitions since deletion of these sequences allowed efficient replication in the presence of the rep gene. The possible role for negative autoregulation of AAV DNA replication in latent infection and helper-dependent replication by AAV is discussed.     

The artificial AT-rich block enhanced the production of bovine growth hormone in Escherichia coli

In order to increase the synthesis of bovine growth hormone (bGH) using T7 promoter system in E. coli, the artificial AT-rich block was introduced into the upstream region of a consensus Shine-Dalgarno (SD) sequence and the spacer region (between SD and ATG codon) was enriched with A and T nucleotides. The cells harboring pTAJ plasmids with AT-rich block produced bGH in the range of 3% to 25% and the cells harboring pTBJ plasmids with AT-rich sequence in the spacer region from 0.8% to 20% of total cell proteins. This result suggests that AT rich block and AT nucleotides in the spacer region destabilize mRNA secondary structure, depending on the downstream coding information of bGH gene and also, implying that the disruption of mRNA secondary structure might be a major factor for regulating bGH expression in the translational initiation process.     

Downregulation of p56(lck) tyrosine kinase activity in T cells of squirrel monkeys (Saimiri sciureus) correlates with the nontransforming and apathogenic properties of herpesvirus saimiri in its natural host

Herpesvirus saimiri is capable of transforming T lymphocytes of various primate species to stable growth in culture. The interaction of the T-cellular tyrosine kinase p56(lck) with the transformation-associated viral protein Tip has been shown before to activate the kinase and provides one model for the T-cell-specific transformation by herpesvirus saimiri subgroup C strains. In contrast to other primate species, squirrel monkeys (Saimiri sciureus) are naturally infected with the virus without signs of lymphoma or other disease. Although the endogenous virus was regularly recovered from peripheral blood cells from squirrel monkeys, we observed that the T cells lost the virus genomes in culture. Superinfection with virus strain C488 did not induce growth transformation, in contrast to parallel experiments with T cells of other primate species. Surprisingly, p56(lck) was enzymatically inactive in primary T-cell lines derived from different squirrel monkeys, although the T cells reacted appropriately to stimulatory signals. The cDNA sequence revealed minor point mutations only, and transfections in COS-7 cells demonstrated that the S. sciureus lck gene codes for a functional enzyme. In S. sciureus, the tyrosine kinase p56(lck) was not activated after T-cell stimulation and enzymatic activity could not be induced by Tip of herpesvirus saimiri C488. However, the suppression of p56(lck) was partially released after administration of the phosphatase inhibitor pervanadate. This argues for unique species-specific conditions in T cells of S. sciureus which may interfere with the transforming activity and pathogenicity of herpesvirus saimiri subgroup C strains in their natural host.     

Herpesvirus saimiri U RNAs are expressed and assembled into ribonucleoprotein particles in the absence of other viral genes.

Marmoset T lymphocytes transformed by herpesvirus saimiri contain a set of five virally encoded U RNAs called HSUR1 through HSUR5. HSUR genes have been individually transfected into a nonlymphoid, nonsimian cell line (HeLa cells) in the absence of any other coding regions of the herpesvirus saimiri genome. The levels of HSUR1 through HSUR4 in HeLa transient-expression systems are comparable to those found in virally transformed T cells (23 to 91%). In contrast, HSUR5 is expressed at ninefold-higher levels in transfected HeLa cells. Immunoprecipitation experiments show that HSURs expressed in transfected cells bind proteins with Sm determinants and acquire a 5' trimethylguanosine cap structure, as they do in transformed T cells. HSUR1 or HSUR4 particles from transfected HeLa cells migrate between 10S and 15S in velocity gradients, identical to the sedimentation of "monoparticles" produced in virally transformed lymphocytes. We conclude from these transfection experiments that no other herpesvirus saimiri or host-cell-specific gene products appear to be required for efficient expression of the HSUR genes or for subsequent assembly of the viral U RNAs into small nuclear ribonucleoprotein particles. In lymphocytes transformed by herpesvirus saimiri, HSUR small nuclear ribonucleoprotein particles are involved in higher-order complexes that sediment between 20S and 25S. HSUR1, HSUR2, and HSUR5 dissociate from such complexes upon incubation at 30 degrees C, whereas the complex containing HSUR4 is stable to incubation.     

New retrovirus helper cells with almost no nucleotide sequence homology to retrovirus vectors.

We prepared retrovirus packaging cell lines containing gag-pol genes from spleen necrosis virus (expressed from a cytomegalovirus promoter and the simian virus 40 (SV40) polyadenylation sequences) and, on a separate vector, either the env gene from spleen necrosis virus (expressed from the Rous sarcoma virus promoter and the SV40 polyadenylation sequences) or the env gene from amphotropic murine leukemia virus (expressed from a cytomegalovirus promoter and the SV40 polyadenylation sequences). The nucleotide sequences in these packaging cell lines have almost no homology to the retrovirus vectors we used. Retrovirus vectors were produced from these new helper cell lines without any genetic interactions between the vectors and sequences in the helper cells and without transfer of the packaging sequences.     

Herpesvirus saimiri vFLIP provides an antiapoptotic function but is not essential for viral replication, transformation, or pathogenicity

Apoptosis of infected cells is an important host defense mechanism, and many viruses have exploited antiapoptotic proteins that interfere with crucial cellular pathways. Viral FLICE inhibitory proteins (vFLIPs) are encoded by rhadinoviruses like herpesvirus saimiri, the related Kaposi's sarcoma-associated herpesvirus-human herpesvirus 8 (KSHV/HHV8), and the poxvirus responsible for molluscum contagiosum. The vFLIPs can block the interaction of the death receptor-adapter complex with the cellular effector FLICE (caspase-8), and this prevents the initiation of the downstream caspase cascade. KSHV/HHV8 vFLIP overexpression can confer resistance to T-cell-mediated apoptosis and acts as a tumor progression factor in a murine B-cell lymphoma model. To analyze the function of herpesvirus vFLIPs in the genetic background of the virus and in a model for viral pathogenesis, we deleted the vFLIP gene (open reading frame 71) from the genome of herpesvirus saimiri strain C488. The viral deletion mutant was viable and replicated like the wild-type virus. An antiapoptotic effect could be attributed to the vFLIP gene, but we also show that the vFLIP gene of herpesvirus saimiri is dispensable for viral transformation of T cells in vitro and for pathogenicity in cottontop tamarins in vivo.     

Metabolic effects of developmental, tissue-, and cell-specific expression of a chimeric phosphoenolpyruvate carboxykinase (GTP)/bovine growth hormone gene in transgenic mice.

Transgenic mice were used to investigate sequences within the promoter of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the rat (EC 4.1.1.32) (PEPCK) which are involved in tissue-specific and developmental regulation of gene expression. Segments of the PEPCK promoter between -2000 and -109 were linked to the structural gene for bovine growth hormone (bGH) and introduced into the germ line of mice by microinjection. Bovine growth hormone mRNA was found in tissues that express the endogenous PEPCK gene, mainly in the liver but to a lesser extent in the kidney, adipose tissue, small intestine, and mammary gland. In the liver the chimeric PEPCK/bGH(460) gene was expressed in periportal cells, which is consistent with the zonation of endogenous PEPCK. The PEPCK/bGH gene was not transcribed in the livers of fetal mice until immediately before birth; at birth the concentration of bGH mRNA increased 200-fold. Our results indicate that the region of the PEPCK promoter from -460 to +73 base pairs contains regulatory sequences required for tissue-specific and developmental regulation of PEPCK gene expression. Mice transgenic for PEPCK/bGH(460) were not hyperglycemic or hyperinsulinemic in response to elevated bGH, as were transgenic mice with the MT/bGH gene. The number of insulin receptors in skeletal muscle was no different in mice transgenic for MT/bGH when compared with mice transgenic for PEPCK/bGH(460) and control animals. However, mRNA abundance for the insulin-sensitive glucose transporter in skeletal muscle was decreased in mice transgenic for the MT/bGH gene. The differences in glucose homeostasis noted with the two types of transgenic mice may be the result of the relative site of expression, the different developmental pattern, or hormonal regulation of expression of the bGH gene.     

Structure and expression of the mRNA for murine granulocyte-macrophage colony stimulating factor.

A cDNA containing a virtually complete copy of the mRNA for the haemopoietic growth regulator, granulocyte-macrophage colony stimulating factor (GM-CSF), has been isolated from a murine T lymphocyte cDNA library. When a eukaryotic expression vector with this cDNA coupled to the SV40 late promoter was introduced into simian COS cells, significant quantities of GM-CSF were secreted. Since all of the biological activities previously ascribed to highly purified GM-CSF were exhibited in the COS cell-derived GM-CSF, all of these activities are intrinsic to the product of a single gene. There are two potential translational initiation codons in the GM-CSF mRNA; the first is buried in the stem and the second located in the loop of a very stable hairpin structure. Expression studies using deletion derivatives of the cDNA indicated that the second AUG is able to initiate the translation and secretion of GM-CSF. The amino acid sequence of the leader peptide is rather atypical for a secreted protein and we speculate that molecules which initiate at the first AUG might exist as integral membrane proteins whereas those initiating at the second are secreted.     

Multihormonal regulation of the human, rat, and bovine growth hormone promoters: differential effects of 3',5'-cyclic adenosine monophosphate, thyroid hormone, and glucocorticoids

We have analyzed the effects of a variety of hormones on activity of the rat GH (rGH), human GH, (hGH), and bovine GH (bGH) promoters. After transient transfection of rat pituitary tumor cells, all three promoters are induced by addition of 8-bromo-cAMP. Sequences required for the cAMP responsiveness of the hGH and rGH promoter lie within 183 base pairs of the mRNA start site. Although the rGH promoter is thyroid hormone (T3) responsive in this system, a construct containing 2.7 kilobases of the hGH promoter 5'-flanking sequences is not. Since we also found that the bGH promoter is T3 responsive in these cells, the hGH results are not likely to be due to a species specific factor required for induction in rat pituitary cells. The hGH promoter is weakly induced by dexamethasone whereas the rGH promoter does not respond to glucocorticoids. The hGH and rGH promoters are not responsive to TRH. These results illustrate the potential heterogeneity in hormonal responses of the same gene in different species.