Monitoring interactions between G-protein-coupled receptors and beta-arrestins.

beta-Arrestins 1 and 2 are ubiquitously expressed intracellular adaptor and scaffolding proteins that play important roles in GPCR (G-protein-coupled receptor) desensitization, internalization, intracellular trafficking and G-protein-independent signalling. Recent developments in BRET (bioluminescence resonance energy transfer) technology enable novel insights to be gained from real-time monitoring of GPCR-beta-arrestin complexes in live cells for prolonged periods. In concert with confocal microscopy, assays for studying internalization and recycling kinetics such as ELISAs, and techniques for measuring downstream signalling pathways such as those involving MAPKs (mitogen-activated protein kinases), investigators can now use a range of experimental tools to elucidate the ever-expanding roles of beta-arrestins in mediating GPCR function.

Bile salt-stimulated lipase (BSSL) distribution in rat, mouse and transgenic mouse expressing human BSSL.

In some species, including man and mouse, bile salt-stimulated lipase (BSSL) in milk catalyzes the hydrolysis of triacylglycerides into glycerol and free fatty acids, a reaction that is of particular importance during suckling. The enzyme is also secreted by the pancreas (referred to as carboxyl-ester hydrolase, CEH). We wished to localize sources and storage sites for BSSL/CEH in rats, in wild-type mice, and in transgenic mice producing recombinant human BSSL in milk. Immunoreactivity against several BSSL fragments was strong in the pancreatic acinar cells and moderate in the absorptive cells of the small intestine and in salivary duct cells of the mice, as well as in rats. Sections from lactating mammary glands of mouse, but not rat, also showed immunoreactivity for BSSL; the signal was strongest in the transgenic mice. Radioactive riboprobes for BSSL mRNA hybridized on sections of rat and mouse pancreatic acinar cells, and mouse mammary glands (both wild-type and transgenic). Using RT-PCR, it was possible to amplify BSSL mRNA from wild-type mouse pancreas and mammary gland, from rat submandibular glands, and, in a few cases, from rat liver. In transgenic mice, the BSSL mRNA was highly expressed only in lactating mammary gland, but could be detected in a few other organs as well.

Production of biologically active salmon calcitonin in the milk of transgenic rabbits.

Salmon calcitonin (sCT) is an example of one of the many bioactive peptides that require amidation of the carboxy terminus for full potency. We describe a method for the production of amidated sCT in the mammary gland of transgenic rabbits. Expression of a fusion protein comprising human alpha lactalbumin joined by an enterokinase cleavable linker to sCT was directed to the mammary gland under the control of the ovine beta lactoglobulin promoter. C-terminal amidation in vivo was achieved by extending the sCT by a single glycine residue that provides a substrate for endogenous amidating activity in the mammary gland. Full characterization of the released sCT demonstrated it to be equivalent to synthetic standard in terms of structure, purity, and potency.

High-level expression of recombinant human fibrinogen in the milk of transgenic mice.

Fibrinogen is a complex plasma protein composed of two each of three different polypeptide chains. We have targeted expression of r-human fibrinogen to the mammary gland of transgenic mice. Three expression cassettes, each containing the genomic sequence for one of the three human fibrinogen chains controlled by sheep whey protein beta-lactoglobulin promoter sequences, were coinjected into fertile mouse eggs. Southern blot analysis demonstrated that more than 80% of the transgenic founders contained all three fibrinogen genes. Reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis of milk from the highest producing founder animal demonstrated the presence of human fibrinogen subunits at concentrations of 2000 micrograms/ml. In several animals with a balanced ratio of the individual fibrinogen subunits, up to 100% of the protein was incorporated into fully assembled fibrinogen hexamers. Incubation of the transgenic milk with thrombin and factor XIII resulted in a cross-linked fibrin clot, indicating that a major portion of the secreted fibrinogen was functional. These studies represent the first report of high-level biosynthesis and secretion of a functional, complex, hexameric protein in the milk of a transgenic animal.

Model for assessing the risk of introducing brucellosis into a brucellosis-free area.

A risk assessment model is presented, for use by local decision-makers to aid the evaluation of proposed changes in existing brucellosis eradication or control programmes. This model provides a format and structure for gathering and analysing data. The model uses data which are generally available and accessible, so that minimum time, expense and effort are required for collection. The use of this model enables an estimation of the risk of introduction of brucellosis into a non-infected population, based on the probability of importing the agent and subsequent spread, given the existence of specified surveillance and control measures. The model creates a point estimate of the risk associated with a given set of conditions.

Transgenic livestock as bioreactors: stable expression of human alpha-1-antitrypsin by a flock of sheep.

We have previously described the generation of transgenic sheep expressing human alpha 1-antitrypsin (h alpha 1AT) in their milk. Here, we report the fidelity of transgene transmission and expression by these animals and their progeny. Transgene transmission has been demonstrated in four of six ovine lines studied. Three of these four lines have exhibited stable transmission of the transgene, whereas the fourth has produced some offspring with reduced copy numbers. Sequential lactations of founder animals has yielded very similar levels of h alpha 1AT protein in milk. Moreover, in one line, derived from a founder male, a flock of seven G1 ewes have yielded comparable levels of h alpha 1AT protein in first and second lactation milk. Two G2 ewes of this line have also produced levels of human protein equivalent to their mother. Although the inheritance of the same transgene in mice was reminiscent of the situation in sheep, stable expression was observed in only one or four lines studied. The importance of these observations to the use of transgenic livestock as bioreactors for the production of human proteins is discussed.

Expression of human alpha 1 antitrypsin in transgenic sheep.

We have recently described the production of large amounts (< or = 65 grams per litre) of enzymatically active human alpha 1 antitrypsin in the milk of transgenic sheep (Wright et al., 1991). Here, we describe in more detail the expression of the human protein in the milk of these animals throughout the lactation period. Human alpha 1 antitrypsin is also found at much lower levels in the plasma of transgenic ewes before, during and after lactation. It is also detected in male plasma at very low levels. We have previously shown human alpha 1 antitrypsin purified from transgenic sheep milk to be indistinguishable from commercially available human plasma derived alpha 1 antitrypsin in terms of gross sugar content and in vitro activity. Here we extend this comparison to more detailed analyses of glycosylation state, amino-terminal sequence, pI value, and molecular weight determination by mass spectrometry.

Mutational analysis of the herpes simplex virus type 1 trans-inducing factor Vmw65.

The herpes simplex virus type 1 (HSV-1) polypeptide Vmw65 is a structural component of the virus particle and is also responsible for trans-induction of immediate early (IE) transcription. Functional domains of this polypeptide were investigated by constructing a series of 10 plasmids each with a 12 bp insertion in the gene encoding Vmw65. Plasmids were analysed for their ability to stimulate IE transcription in short term transfection assays, and the altered Vmw65 polypeptides were assayed for the ability to form an IE-specific protein-DNA complex (IEC) in vitro. A direct correlation was observed between stimulation of transcription and formation of IEC, strongly suggesting that IEC is an important intermediate in transcription activation. Plasmids were also tested for their ability to rescue the temperature-sensitive mutation in the HSV-2 assembly mutant ts2203, since marker rescue analysis indicated that this mutation maps within the gene encoding Vmw65. Five plasmids failed to rescue ts2203, thereby defining regions of Vmw65 required for virus assembly. The results show that distinct domains exist in Vmw65 for activation of transcription and assembly of virus.

The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1.

We have determined the DNA sequence of the long unique region (UL) in the genome of herpes simplex virus type 1 (HSV-1) strain 17. The UL sequence contained 107,943 residues and had a base composition of 66.9% G + C. Together with our previous work, this completes the sequence of HSV-1 DNA, giving a total genome length of 152,260 residues of base composition 68.3% G + C. Genes in the UL region were located by the use of published mapping analyses, transcript structures and sequence data, and by examination of DNA sequence characteristics. Fifty-six genes were identified, accounting for most of the sequence. Some 28 of these are at present of unknown function. The gene layout for UL was found to be very similar to that for the corresponding part of the genome of varicella-zoster virus, the only other completely sequenced alphaherpesvirus, and the amino acid sequences of equivalent proteins showed a range of similarities. In the whole genome of HSV-1 we now recognize 72 genes which encode 70 distinct proteins.

Structures of herpes simplex virus type 1 genes required for replication of virus DNA.

Recently, a method has been developed to identify regions in the genome of herpes simplex virus type 1 (HSV-1) which contain genes required for DNA synthesis from an HSV-1 origin of DNA replication, and seven genomic loci have been identified as representing the necessary and sufficient gene set for such replication (C. A. Wu, N. J. Nelson, D. J. McGeoch, and M. D. Challberg, J. Virol. 62:435-443, 1988). Two of the loci represent the well-known genes for DNA polymerase and major DNA-binding protein, but the remainder had little or no previous characterization. In this report we present the DNA sequences of the five newly identified genes and their deduced transcript organizations and encoded amino acid sequences. These genes were designated UL5, UL8, UL9, UL42, and UL52 and were predicted to encode proteins with molecular weights of, respectively, 99,000, 80,000, 94,000, 51,000, and 114,000. All of these genes had clear counterparts in the genome of the related alphaherpesvirus varicella-zoster virus, but only UL5 and UL52 were detectably conserved in the distantly related gammaherpesvirus Epstein-Barr virus, as judged by amino acid sequence similarity. The sequence of the UL5 protein, and of its counterparts in the other viruses, contained a region closely resembling known ATP-binding sites; this could be indicative, for instance, of a helicase or primase activity.

DNA sequence of the herpes simplex virus type 1 gene whose product is responsible for transcriptional activation of immediate early promoters.

Previous work has shown that transcriptional activation of herpes simplex virus type 1 (HSV-1) immediate early genes is mediated by a protein species (Vmw65) present in the tegument of infecting virions. This paper describes DNA sequence analysis and mRNA mapping of the Vmw65 gene in HSV-1 strain 17. The Vmw65 coding region was identified as a 490 codon sequence encoding a polypeptide of molecular weight 54,342 and characterised by a high proportion of charged amino acid residues. A homologue to Vmw65 was detected in the genome of varicella-zoster virus, another human herpesvirus. Apart from its role in trans-activation, Vmw65 is a major constituent of the virion. Its possible significance in virus structure is discussed.

Reservoir animals for nephropathia epidemica in Norway: indications of a major role for the bank vole (C. glareolus) in comparison with the woodmouse (A. sylvaticus).

Small rodents were collected live in two different locations within a nephropathia epidemica (NE) endemic area, and tested for both antiviral serum antibodies and viral antigens in lung sections. In one location, only Apodemus sylvaticus (woodmice) were found in the traps, in the other, both A. sylvaticus and Clethrionomys glareolus (bank voles) were collected. Among the woodmice from the former location the prevalence of NE virus markers was significantly lower than for either woodmice or bank voles from the other location, and no NE antigen-positive animals was found. The woodmice co-existing with bank voles had a lower prevalence of NE antigen and antibodies than the bank voles, and fewer woodmice had both antibodies and antigen. The results emphasize the important role of bank voles as a major NE virus reservoir and probable source of human infections.