Mutations in the E1a gene of type 5 adenovirus result in oncogenic transformation of Fischer rat embryo cells

Transformation of a specific clone of Fischer rat embryo (CREF) cells with wild-type 5 adenovirus (Ad5) or the E1a plus E1b transforming gene regions of Ad5 results in epithelioid transformants that grow efficiently in agar but that do not induce tumors when inoculated into nude mice or syngeneic Fischer rats. In contrast, CREF cells transformed by a host-range Ad5 mutant, H5hrl, which contains a single base-pair deletion of nucleotide 1055 in E1a resulting in a 28-kd protein (calculated) in place of the wild-type 51-kd acidic protein, display a cold-sensitive transformation phenotype and an incomplete fibroblastic morphology but surprisingly do induce tumors in nude mice and syngeneic rats. Tumors develop in both types of animals following injection of CREF cells transformed by other cold-sensitive Ad5 E1a mutants (H5dl101 and H5in106), which contain alterations in their 13S mRNA and consequently truncated 289AA proteins. CREF cells transformed with only the E1a gene (0-4.5 m.u.) from H5hrl or H5dl101 also produce tumors in these animals. To directly determine the role of the 13S E1a encoded 289AA protein and the 12S E1a encoded 243AA protein in initiating an oncogenic phenotype in adenovirus-transformed CREF cells, we generated transformed cell lines following infection with the Ad2 mutant pm975, which synthesizes the 289AA E1a protein but not the 243AA protein, and the Ad5 mutant H5dl520 and the Ad2 mutant H2dl1500, which do not produce the 289AA E1a protein but synthesize the normal 243AA E1a protein. All three types of mutant adenovirus-transformed CREF cells induced tumors in nude mice and syngeneic rats. Tumor formation by these mutant adenovirus-transformed CREF cells was not associated with changes in the arrangement of integrated adenovirus DNA or in the expression of adenovirus early genes. These results indicate, therefore, that oncogenic transformation of CREF cells can occur in the presence of a wild-type 13S E1a protein or a wild-type 12S E1a protein when either protein is present alone, but does not occur when both wild-type E1a proteins are present.     

Two Lyt-2 polypeptides arise from a single gene by alternative splicing patterns of mRNA

The Lyt-2/3 molecule is a glycoprotein expressed on T lymphocytes and has classically been considered a marker for the cytotoxic/suppressor T cell subset. It has been postulated to be a receptor for class I major histocompatibility complex proteins. We have used a cDNA clone encoding the analogous human protein, Leu-2/T8, to isolate mouse cDNA clones, which were used as probes to isolate mouse genomic clones. By transfection we have shown that the mouse homologue of Leu-2/T8 is Lyt-2 and not Lyt-3. We have further demonstrated that two Lyt-2 polypeptide chains are encoded by a single gene and result from alternative modes of mRNA splicing. The nucleotide sequence of cDNA clones encoding each of these polypeptide chains has been determined and shows the difference between the two Lyt-2 polypeptide chains to be in the lengths of their cytoplasmic tails.     

A comparative description of mitochondrial DNA differentiation in selected avian and other vertebrate genera

Levels of mitochondrial DNA (mtDNA) sequence divergence between species within each of several avian (Anas, Aythya, Dendroica, Melospiza, and Zonotrichia) and nonavian (Lepomis and Hyla) vertebrate genera were compared. An analysis of digestion profiles generated by 13-18 restriction endonucleases indicates little overlap in magnitude of mtDNA divergence for the avian versus nonavian taxa examined. In 55 interspecific comparisons among the avian congeners, the fraction of identical fragment lengths (F) ranged from 0.26 to 0.96 (F = 0.46), and, given certain assumptions, these translate into estimates of nucleotide sequence divergence (p) ranging from 0.007 to 0.088; in 46 comparisons among the fish and amphibian congeners, F values ranged from 0.00 to 0.36 (F = 0.09), yielding estimates of P greater than 0.070. The small mtDNA distances among avian congeners are associated with protein-electrophoretic distances (D values) less than approximately 0.2, while the mtDNA distances among assayed fish and amphibian congeners are associated with D values usually greater than 0.4. Since the conservative pattern of protein differentiation previously reported for many avian versus nonavian taxa now appears to be paralleled by a conservative pattern of mtDNA divergence, it seems increasingly likely that many avian species have shared more recent common ancestors than have their nonavian taxonomic counterparts. However, estimates of avian divergence times derived from mtDNA- and protein-calibrated clocks cannot readily be reconciled with some published dates based on limited fossil remains. If the earlier paleontological interpretations are valid, then protein and mtDNA evolution must be somewhat decelerated in birds. The empirical and conceptual issues raised by these findings are highly analogous to those in the long-standing debate about rates of molecular evolution and times of separation of ancestral hominids from African apes.      

Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from humans and apes

Statistical methods for computing the standard errors of the branching points of an evolutionary tree are developed. These methods are for the unweighted pair-group method-determined (UPGMA) trees reconstructed from molecular data such as amino acid sequences, nucleotide sequences, restriction-sites data, and electrophoretic distances. They were applied to data for the human, chimpanzee, gorilla, orangutan, and gibbon species. Among the four different sets of data used, DNA sequences for an 895-nucleotide segment of mitochondrial DNA (Brown et al. 1982) gave the most reliable tree, whereas electrophoretic data (Bruce and Ayala 1979) gave the least reliable one. The DNA sequence data suggested that the chimpanzee is the closest and that the gorilla is the next closest to the human species. The orangutan and gibbon are more distantly related to man than is the gorilla. This topology of the tree is in agreement with that for the tree obtained from chromosomal studies and DNA-hybridization experiments. However, the difference between the branching point for the human and the chimpanzee species and that for the gorilla species and the human-chimpanzee group is not statistically significant. In addition to this analysis, various factors that affect the accuracy of an estimated tree are discussed.      

Trace elements and lipid peroxidation in uremic patients on hemodialysis

Trace elements and lipid peroxidation in 26 patients with chronic renal failure treated with hemodialysis and 25 healthy subjects were observed. Both plasma and erythrocyte trace elements and plasma malon dialdehyde (MDA) were examined immediately before and after hemodialysis. Increased levels of plasma Cu, MDA, and erythrocyte Pb, Mn, Zn, and a significantly decreased plasma Se, Zn and erythrocyte Se were found in patients before hemodialysis. After a single hemodialysis, erythrocyte Mn, Cu, Zn, and plasma Cu, Al, and MDA were significantly increased whereas both plasma and erythrocyte Se were lower in patients than in healthy subjects. The level of MDA was not significantly changed during the single hemodialysis. Both plasma and erythrocyte Zn levels and plasma Cu and Al were significantly higher after hemodialysis than before hemodialysis. In conclusion, levels of trace elements are altered by hemodialysis, which may increase patient susceptibility to lipid peroxidation in uremia.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

Of mice and Marfan: genetic linkage analyses of the fibrillin genes,Fbn1 and Fbn2, in the mouse genome

The fibrillin genes, FBN1 and FBN2, encode large extracellular matrix glycoproteins involved in the structure and function of microfibrils. Mutations in FBN1 are found in patients with Marfan syndrome, a heritable connective tissue disease that primarily affects the cardiovascular, ocular, and skeletal systems. We extended the studies of these genes by determining their chromosomal position in the mouse genome. Restriction fragment length polymorphisms (RFLPs) between the progenitors of an interspecific backcross involving AEJ/Gn and Mus spretus mice were used to establish the segregation patterns of the murine homologs, Fbn1 and Fbn2, in the backcross progeny. The results position Fbn1 between the B2m and Illa genes on mouse Chromosome (Chr) 2 and establish its candidacy for the Tight skin (Tsk) mutation. The results position Fbn2 between the D18Mit35 and Pdgfrb loci in the central region of mouse Chr 18. Fbn2 maps near three mutations [bouncy (bc), plucked (pk), and shaker with syndactyly (sy)] and may be a candidate for the pk mutation.