overexpression. In contrast, secretion of M-CSF, which was not detected in association with GRP78, was unaffected. This indicates that elevated levels of GRP78 may increase stable association and decrease the secretion efficiency of proteins which normally transiently associate with GRP78. These results indicate that one function of GRP78 is selective protein retention in the ER.

Factor VIII (antihemophilic factor) is a high molecular weight plasma glycoprotein that participates in the blood clotting cascade. The recent cloning and sequence analysis of the cDNA encoding human factor VIII revealed an obvious domain structure for the protein, which can be represented as Al-A2-B-A3-Cl-C2. We now report the DNA sequence analysis of porcine exons encoding the entire B domain and part of the A2 and A3 domains. We found an unusually high degree of porcine-human amino acid sequence divergence in the B region compared with the limited sequence available for other regions of the porcine factor VIII molecule. In addition to sequence divergence, there are numerous gaps in the porcine B domain totalling over 200 amino acids. Recombinant DNA techniques were used to effect the removal of large segments of DNA encoding the B domain from the full-length human factor VIII cDNA. These constructs directed the synthesis of biologically active factor VIII when introduced into mammalian cells despite the deletion of up to 38% ofthe factor VIII molecule.Hemophilia A is a bleeding disorder resulting from a deficiency or abnormality of the blood clotting protein, factor VIII (for review, see ref. 1). Factor VIII functions in the blood clotting cascade as the cofactor for factor IXa proteolytic activation offactor X. The blood clotting pathway in which factor VIII participates eventually results in the proteolytic cleavage of fibrinogen to form insoluble fibrin polymers. In vivo, fibrin deposition in conjunction with platelet aggregation act to curtail blood loss from a damaged vessel.The cofactor activity of factor VIII acts to increase the Vm,, of the factor IXa-dependent activation of factor X by at least 4 orders of magnitude. Factor VIII does not function proteolytically in this reaction but can itself be proteolytically activated by other coagulation enzymes such as factor X or thrombin. Neither the mechanism of factor VIII activation nor the nature of its cofactor activity is well understood.Recently the entire human factor VIII gene, spanning over 185 kilobases (kb) of the X chromosome, and full-length cDNA have been cloned (2-5). The DNA sequence of the cDNA and NH2-terminal amino acid sequence analysis of the plasma protein shows factor VIII to be synthesized as a single-chain precursor of 2351 amino acids from which a 19-amino acid "signal sequence" is cleaved during translation. A computer-aided search for factor VIII intramolecular homologies revealed three distinct structural domains, including a triplicated region (A domain) of -330 amino acids, a duplicated region (C domain) of '=150 amino acids, and a unique region (B domain) of -980 amino acids. These domains are arranged in the order A1-A2-B-A3-C1-C2 (see In this report, we present DNA sequence analysis that demonstrates a very high degree of divergence between porcine and human B domains of the factor VIII gene. In addition, removal of the majority of DNA encoding the B domain from a full-length human factor VIII cDNA is not detrimental...

Dicistronic mRNA expression vectors efficiently translate a 5' open reading frame (ORF) and contain a selectable marker within the 3' end which is inefficiently translated. In these vectors, the efficiency of translation of the selectable 3' ORF is reduced approximately 100-fold and is highly dependent on the particular sequences inserted into the 5' cloning site. Upon selection for expression of the selection marker gene product, deletions within the 5' ORF occur to yield more efficient translation of the selectable marker. We have generated improved dicistronic mRNA expression vectors by utilization of a putative internal ribosomal entry site isolated from encephalomyocarditis (EMC) virus. Insertion of the EMC virus leader sequence upstream of an ORF encoding either a wildtype or methotrexate resistant dihydrofolate reductase (DHFR) reduces DHFR translation up to 10-fold in a monocistronic DHFR expression vector. However, insertion of another ORF upstream of the EMC leader to produce a dicistronic mRNA does not further reduce DHFR translation. In the presence of the EMC virus leader, DHFR translation is not dependent on sequences inserted into the 5' end of the mRNA. We demonstrate that stable high level expression of inserted cDNAs may be rapidly achieved by selection for methotrexate resistance in DHFR deficient as well as DHFR containing cells. In contrast to previously described dicistronic expression vectors, these new vectors do not undergo rearrangement or deletion upon selection for amplification by propagation in increasing concentrations of methotrexate. The explanation may be either that the EMC virus leader sequence allows internal initiation of translation or that cryptic splice sites in the EMC virus sequence mediate production of monocistronic mRNAs. These vectors may be generally useful to rapidly obtain high level expression of cDNA genes in mammalian cells.

In plasma, antihemophilic factor (factor VIII) exists as a 200-kilodalton heavy-chain polypeptide in a metal ion association with an 80-kilodalton light-chain polypeptide. This complex is bound by hydrophobic and hydrophilic interactions to a large multimeric glycoprotein, von Willebrand factor (vWF). Accumulation of secreted human factor VIII activity expressed in Chinese hamster ovary cells requires the addition of serum in the growth medium, which provides vWF. Here we report that coexpression of vWF with factor VIII in Chinese hamster ovary cells resulted in increased stable accumulation of factor VIII activity in the absence of serum in the growth medium. In the coexpressing cells, the vWF cDNA transcription unit was transcribed to yield mRNA which was efficiently translated. vWF was properly processed and secreted to yield disulfide-bonded high-molecular-weight multimers similar to those observed in vWF secreted from human endothelial cells. Nuclear run-on assays showed that the factor VIII gene was transcribed at a level similar to that of the vWF gene, but the mRNA did not accumulate to high levels in the cytoplasm. In addition, although the translation efficiency of the factor VIII mRNA was similar to that of vWF, the processing and secretion of the factor VIII primary translation product was dramatically reduced compared with vWF. These results demonstrate that in Chinese hamster ovary cells both factor VIII mRNA accumulation and the processing and secretion of the primary factor VIII translation product are inefficient processes.The factor VIII complex has two distinct biologic functions: coagulant activity and a role in primary hemostasis (for reviews, see references 32 and 43). The analysis of factor VIII deficiency diseases, classic hemophilia and von Willebrand's disease, have contributed to the understanding that factor VIII is a complex of two components: the factor VIII procoagulant protein (antihemophilic factor) and the factor VIII-related antigen (von Willebrand factor [vWF]).The factor VIII molecule is an important regulatory protein in the blood coagulation cascade. After activation by thrombin, it accelerates the rate of factor X activation by factor IXa, eventually leading to the formation of the fibrin clot (18,28,39,49,50). Factor VIII is synthesized as a large precursor which is cleaved to generate an amino-terminalderived heavy chain of 200 kilodaltons (kDa) in a metal ion-stabilized complex with the carboxy-terminal-derived light chain of 80 kDa (9,13,41,51). Factor VIII has binding sites for factor IXa, factor X, Ca2 , phospholipid, and vWF.

Expression of human tissue-type plasminogen activator (t-PA) at high levels has been achieved in Chinese hamster ovary (CHO) cells by cotransfection and subsequent coampiffication of the transfected sequences. Expression vectors containing the t-PA cDNA gene and a dihydrofolate reductase (DHFR) cDNA gene were cotransfected into CHO DHFR-deficient cells. Transformants expressing DHFR were selected by growth in media lacking nucleosides and contained low numbers of t-PA genes and DHFR genes. Stepwise selection of the DHFR+ transformants in increasing concentrations of methotrexate generated cells which had amplified both DHFR genes and t-PA genes over 100-fold. These cell lines expressed elevated levels of enzymatically active t-PA. To optimize both t-PA sequence amplification and t-PA expression, various modifications of the original procedure were used. These included alterations to the DHFR expression vector, optimization of the molar ratio of t-PA to DHFR sequences in the cotransfection, and modification of the methotrexate resistance selection procedure. The structure of the amplified DNA, its chromosomal location, and its stability during growth in the absence of methotrexate are reported.