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.

Recognition of the adenovirus type 2 origin of DNA replication by the virally encoded DNA polymerase and preterminal proteins.

Initiation of adenovirus DNA synthesis is preceded by the assembly of a nucleoprotein complex at the origin of DNA replication containing three viral proteins, preterminal protein, DNA polymerase and DNA binding protein, and two cellular proteins, nuclear factors I and III. While sequence specific interactions of the cellular proteins with their cognate sites in the origin of DNA replication are well characterized, the question of how the viral replication proteins recognize the origin has remained unanswered. Preterminal protein and DNA polymerase were therefore purified to homogeneity from recombinant baculovirus infected insect cells. Gel filtration demonstrated that while DNA polymerase existed in monomeric and dimeric forms, preterminal protein was predominantly monomeric and when combined the proteins formed a stable heterodimer. In a gel electrophoresis DNA binding assay each of the protein species recognized DNA within the origin of DNA replication with unique specificity. Competition analysis and DNase I protection experiments revealed that although each protein could recognize the origin, the heterodimer did so with enhanced specificity, protecting bases 8-17 from cleavage with the nuclease. Thus the highly conserved 'core' of the origin of DNA replication, present in all human adenoviruses, is recognized by the preterminal protein--DNA polymerase heterodimer.

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.

The DNA-binding domain of nuclear factor I is sufficient to cooperate with the adenovirus type 2 DNA-binding protein in viral DNA replication.

Recombinant baculoviruses have been constructed which express the full-length nuclear factor I (NFI) protein or a derivative of NFI that contains only the DNA-binding domain of the protein in infected insect cells. Both proteins were purified from insect cells infected with the respective baculoviruses and tested for their ability to cooperate with the adenovirus type 2 (Ad2) DNA-binding protein during virus replication. DNase I protection experiments demonstrated that the viral DNA-binding protein increased the affinity of both the full-length NFI and the DNA-binding domain of NFI for their recognition site in the Ad2 origin of DNA replication. As a consequence, the NFI-dependent increase in the efficiency of DNA replication observed upon addition of viral DNA-binding protein was the same when the full-length or DNA-binding domain derivative of NFI was added. Thus it appears that all of the activities associated with the ability of NFI to stimulate Ad2 DNA replication are located within the DNA-binding domain of the protein.

Identification of two distinct regions within the adenovirus minimal origin of replication that are required for adenovirus type 4 DNA replication in vitro.

The adenovirus type 4 origins of replication are located at each end of the linear, protein-linked viral DNA molecule and consist of the terminal 18 bp of the viral genome. The sequence of the first 8 bp of the viral genome varies among different adenovirus serotypes, but the sequence from bp 9 to 18 is conserved in all human serotypes, suggesting that it may be of critical importance to origin function. Using an in vitro system in which purified fractions or crude extracts of adenovirus type 4-infected HeLa cells can support initiation and elongation on linearized plasmid templates containing cloned origin sequences, we examined the effect of single base changes in positions 9 to 18 of the adenovirus origin on DNA replication in vitro. Changes in positions 12 to 16 have little effect, whereas alterations at positions 9, 10, 11, 17, and 18 all reduce the efficiency of initiation of DNA replication by between 50 and 90%. Our results show that the region from bp 9 to 18 contains two sets of bases essential for DNA replication which are separated by 5 bp in which single base changes can be accommodated. The likely role of the region from bp 9 to 18 as containing the recognition sequence for a DNA-protein interaction essential for viral DNA replication is discussed.

Replication of adenovirus type 4 DNA by a purified fraction from infected cells.

An extract from Adenovirus type 4 infected HeLa cells was fractionated by ion-exchange and DNA affinity chromatography. One fraction, which bound tightly to single stranded DNA, contained predominantly a protein of apparent molecular weight 65,000 and three less abundant proteins. Immunological cross-reactivity with adenovirus type 2 proteins confirmed the presence of preterminal protein and indicated that the abundant species was the virus coded DNA binding protein. This fraction contained an aphidicolin resistant DNA polymerase activity and in the presence of a linearised plasmid containing the adenovirus type 4 origin of DNA replication efficient transfer of dCMP onto preterminal protein, indicative of initiation, was observed. Furthermore, addition of all four deoxyribonucleotide triphosphates and an ATP regenerating system resulted in the elongation of initiated molecules to generate plasmid molecules covalently attached to preterminal protein. Adenovirus type 4 DNA binding protein was extensively purified from crude adenovirus-4 infected HeLa extract by immunoaffinity chromatography using a monoclonal antibody raised against adenovirus type 2 DNA binding protein. A low level of initiation of DNA replication was detected in the fraction depleted of DNA binding protein but activity was restored by addition of purified DNA binding protein. DNA binding protein therefore plays an important role in the initiation of Ad4 DNA replication.

A distinct octamer-binding protein present in malignant melanoma cells.

The octamer-binding proteins present in HeLa cells, B-cells and malignant melanoma cells were compared by a gel-electrophoresis DNA-binding assay. Using an extract from the malignant melanoma cells a complex was formed using a variety of octamer containing probes that was distinct from those found using either a HeLa or B-cell extract. DNAase 1 footprints and methylation interference patterns of the melanoma-specific octamer-binding protein were indistinguishable from those obtained with the HeLa factor NF-A1, except for preferential binding of the melanoma-specific factor to DNA methylated at two G residues 16 base-pairs 3' to the octamer motif. Competition analyses using a variety of wild-type and mutant probes showed that mutations affecting binding of NF-A1 similarly affected binding of the melanoma octamer-binding factor. These data also revealed the extreme flexibility of the octamer-binding site, with one probe sharing only 4 bases with the 8 base consensus sequence binding efficiently.

Multiple transcription factors interact with the adenovirus-2 EII-late promoter: evidence for a novel CCAAT recognition factor.

Multiple cellular transcription factors have been shown to interact with the upstream region of the adenovirus-2 EIIa-late promoter. One of these factors recognises each of the three CCAAT motifs present in the EIIL promoter at positions -72, -135 and -229, as well as the CCAAT elements in the rat albumin and herpes virus thymidine kinase promoters. A mutation known to reduce thymidine kinase promoter activity in vivo and in vitro abolishes binding of the factor, termed CCAAT recognition factor (CRF), which appears to be distinct from previously identified CCAAT factors. In addition, another protein, termed upstream factor II (USFII), shares binding sites at position -110 in the EIIL promoter and in the c-fos enhancer adjacent to the serum regulatable element. The recognition site for USFII is also found in the c-fos promoter and in the adenovirus early region EIV and EIIa-early promoters. An Sp1 recognition site has also been identified at position -41, and the binding sites for Sp1, USFII and CRF are all required for efficient EIIa-late promoter function. Finally, an additional factor recognising the consensus GGGGGGNT has been detected.

Reserpic acid as an inhibitor of norepinephrine transport into chromaffin vesicle ghosts.

Reserpic acid, a derivative of the antihypertensive drug reserpine, inhibits catecholamine transport into adrenal medullary chromaffin vesicles. Since it does not affect the membrane potential generated by the H+-translocating adenosine triphosphatase but inhibits ATP-dependent norepinephrine uptake with a Ki of about 10 microM, reserpic acid must block the H+/monoamine translocator. Because reserpic acid is much more polar than reserpine, it does not permeate the chromaffin vesicle membrane, nor is it transported into chromaffin vesicle ghosts in the presence of Mg2+-ATP. Although it inhibits norepinephrine transport when added externally, reserpic acid does not inhibit when trapped inside chromaffin vesicle ghosts. Therefore, reserpic acid must bind to the external face of the monoamine translocator and should be a good probe of the translocator's structural asymmetry.