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Analysis of several mutations in the mouse is providing useful insights into the nature of the genes required for the establishment of the left-right axis during early development. Here we describe a new targeted allele of the mouse Tg737 gene, Tg737(Delta)2-3(beta)Gal), which causes defects in left-right asymmetry and other abnormalities during embryogenesis. The Tg737 gene was originally identified based on its association with the mouse Oak Ridge Polycystic Kidney (orpk) insertional mutation, which causes polycystic kidney disease and other defects. Complementation tests between the original orpk mutation and the new targeted knock-out mutation demonstrate that Tg737(Delta)2-3(beta)Gal) behaves as an allele of Tg737. The differences in the phenotype between the two mutations suggest that the orpk mutation is a hypomorphic allele of the Tg737 gene. Unlike the orpk allele, where all homozygotes survive to birth, embryos homozygous for the Tg737(Delta)2-3(beta)Gal) mutation arrest in development at mid-gestation and exhibit neural tube defects, enlargement of the pericardial sac and, most notably, left-right asymmetry defects. At mid-gestation the direction of heart looping is randomized, and at earlier stages in development lefty-2 and nodal, which are normally expressed asymmetrically, exhibit symmetrical expression in the mutant embryos. Additionally, we determined that the ventral node cells in mutant embryos fail to express the central cilium, which is a characteristic and potentially functional feature of these cells. The expression of both Shh and Hnf3(beta) is downregulated in the midline at E8.0, indicating that there are significant alterations in midline development in the Tg737(Delta)2-3(beta)Gal) homozygous embryos. We propose that the failure of ventral node cells to fully mature alters their ability to undergo differentiation as they migrate out of the node to contribute to the developing midline structures. Analysis of this new knockout allele allows us to define a critical role for the Tg737 gene during early embryogenesis. We have named the product of the Tg737 gene Polaris, which is based on the various polarity related defects associated with the different alleles of the Tg737 gene.

Mouse liver stem cell (oval cell) lines were investigated in order to determine the role which two families of growth and differentiation factors (GDFs), epidermal growth factor (EGF) family and transforming growth factor beta (TGF-beta) family, play in liver regeneration. EGF family members, including EGF, amphiregulin, betacellulin, heparin-binding epidermal growth factor, and TGF-alpha, were mitogenic for oval cell lines while TGF-beta family members, including TGF-beta1, TGF-beta2 and TGF-beta3, inhibited mitogenesis and induced apoptosis in oval cell lines. Surprisingly, the combination of EGF family members and TGF-ss family members resulted in neither proliferation nor apoptosis but instead in a novel cellular response, cellular scattering in tissue culture and morphological differentiation in Matrigel. Analysis of the signal transduction pathways activated by exposure of oval cell lines to either EGF, EGF+TGF-beta, or TGF-beta indicated that novel combinations of intracellular signals result following stimulation of the cells with the combination of EGF+TGF-beta. These data reveal that the dynamics of synergistic GDF action following tissue injury and regeneration results in a new level of complexity not obvious from the study of individual GDFs.

We previously reported that papillomas can arise from the follicular epithelium of v-Ha-ras transgenic TG.AC mice. Since the viable-yellow mutation (A^vy) of the mouse agouti gene which regulates coat color pigmentation by acting within the micro-environment of the hair follicle has been shown to function as a tumor promoter in the liver, we hypothesized that it may also play a role in TG.AC skin tumorigenesis. Endogenous agouti protein product was detected in the outer root sheath of anagen hair follicles following plucking of the hair shaft, but not in the interfollicular epithelium, in TG.AC mice on an FVB/N genetic background. It was also detected in papillomas from these mice produced by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment or plucking. Expression of the A^vy allele in the v-Ha-ras transgenic TG.AC mouse line results in an 2-fold increase in papilloma development compared with controls which did not carry the A^vy allele following twice-weekly treatment with 1.25, 2.5 or 5.0 μg TPA. In addition, TPA-treated, papilloma-bearing F₁ mice which carried the A^vy allele, but not F₁ mice which did not carry the A^vy allele, exhibited a syndrome of humoral hypercalcemia mediated by parathyroid hormone-related protein (PTHrP) that led to weight loss, hypercalcemia and hypophosphatemia. Thus, we conclude that the A^vy allele can influence the development of skin tumors and PTHrP-mediated humoral hypercalcemia in v-Ha-ras transgenic TG.AC mice.

RNA polymerase from cells of Escherichia coli infected with T5 were recovered as a complex with two pre-early phage-coded polypeptides, the 60,000-dalton product of gene A1 and a previously reported 11,000-dalton polypeptide. This RNA polymerase complex had altered transcriptional specificity, in that it transcribed pre-early genes less efficiently than it did early genes.

A cDNA clone for the pre-alpha subunit of the pituitary glycoprotein hormones has been isolated from a bovine pituitary cDNA library through the use of a pool of synthetic oligodeoxynucleotide probes. This clone, designated pB alpha, contains a 564-base pair insert which includes a portion of the signal sequence, the entire coding sequence of the mature protein, and 224 base pairs of the 3'-untranslated sequence. As expected, the nucleotide and amino acid sequence of the mature bovine alpha subunit was homologous to the sequences reported for humans and rodents, with the most extensive homology occurring between bovine and rodents (85-90%). However, a comparison of the 3'-untranslated regions of pre-alpha subunit mRNA from three different mammalian species indicated that in bovine and rat, or in human and rat, these sequences have rapidly diverged, yielding respective homologies of 21 and 36%. In contrast, the sequence homology observed between the 3'-untranslated regions of bovine and human was 79%, which approaches the level of homology shared by their coding sequences. Thus, the conservation of the 3'-untranslated sequence in bovine and human pre-alpha subunit mRNA may be an indication that this region is functionally significant in these two species.

A strain of herpesvirus saimiri containing a bovine growth hormone (bGH) gene under the control of the simian virus 40 (SV40) late-region promoter was constructed. This strain, bGH-Z20, was replication competent and stably harbored the bGH gene upon serial passage. Nonpermissive marmoset T cells persistently infected with bGH-Z20 produced a 0.9-kilobase RNA which contained all of the bGH exon sequences and appeared to initiate within the SV40 promoter region. However, in permissively infected owl monkey kidney cells, RNAs containing growth hormone sequences appeared to initiate from herpesvirus saimiri promoters positioned upstream from the SV40-growth hormone gene. Persistently infected T cells in culture secreted 500 ng of bGH protein per 10(6) cells per 24 h during the several months of testing. The secreted protein was 21 kilodaltons, the size of authentic bGH. New World primates experimentally infected with bGH-Z20 produced circulating bGH and developed immunoglobulin G antibodies directed against bGH. Because herpesviruses characteristically remain latent in the infected host, these observations suggest a means for replacing gene products missing or defective in hereditary genetic disorders.

The poly(A) site of bovine prolactin (bPRL) mRNA was examined by phased priming of cDNA synthesis with oligodeoxynucleotides of the general sequence d(pT8-N-N'). The existence of multiple poly(A)-adjacent sequences in bPRL mRNA was indicated by the production of specific chain-termination fragments with at least three d(pT8-N-N') sequences. Comparison of the sequence bands produced by initiation of cDNA synthesis on the bPRL mRNA template with d(pT8-A-G), d(pT8-G-A), and d(pT8-C-G) revealed a shifted pattern of identical fragments. The shift in position of related sequence bands on the gel suggested that the difference in length of the three major bPRL mRNA species occurred within a span of 12 nucleotides. Sequence analysis conducted with the three d(pT8-N-N') primers gave identical nucleotide sequences for the 3' noncoding region of the bPRL mRNA species and suggested that the mRNA molecules were heterogeneous in length. The existence of multiple poly(A) sites was confirmed by determination of the nucleotide sequence of bPRL cDNA clones containing two of the major poly(A)-adjacent sequences predicted by the oligodeoxynucleotide primers. The mRNA molecules containing these multiple poly(A)-addition sites were shown to be present in the bPRL mRNA obtained from a single pituitary gland. The variation in the poly(A) junction of bPRL mRNA may be a reflection of the processing events at the 3' terminus of mRNAs.

We examined whether the sequence extending 3' to the polyadenylylation site of the bovine growth hormone gene contains any signal that affects the polyadenylylation of the growth hormone mRNA. For this purpose, cloned copies of this gene, each containing a different length of growth hormone-specific sequence 3' to the wild-type polyadenylylation site, were used to transfect COS-1 cells. The polyadenylylation site on the mRNAs produced from the exogenously added growth hormone genes were analyzed with an S1 nuclease mapping procedure. We found that a gene containing 84 base pairs of its own 3' flanking sequence is capable of producing an accurately polyadenylylated mRNA. On the other hand, genes containing only 1, 10, or 13 base pairs of 3' flanking sequence were principally polyadenylylated at discrete sites either upstream or downstream from the wild-type position. Using a computer program, we examined whether secondary structures on the primary growth hormone transcript correlated with the site where the mRNA is polyadenylylated.