Regulation of albumin gene expression in a series of rat hepatocyte cell lines immortalized by simian virus 40 and maintained in chemically defined medium.

A series of simian virus 40 (SV40)-immortalized hepatocyte cell lines were characterized for albumin production, the regulation of albumin production, and the expression of other liver-specific genes. This series of cell lines is particularly useful for studying the regulation of hepatocyte gene expression because the cell lines express liverlike levels of a number of liver-specific functions and do so while growing in a chemically defined medium. SV40-immortalized hepatocyte cell lines were derived from colonies of albumin-producing epithelial cells that arose after primary hepatocytes maintained in chemically defined medium were transfected with SV40 DNA. Some cell lines secreted albumin at levels equal to or greater than those secreted by freshly plated primary hepatocytes, and all but one line continued to produce albumin for more than 20 passages. The variation in albumin secretion among cell lines reflected differences in the amount of albumin produced per cell and not in the percentage of albumin-producing cells in each line. The characterization of selected cell lines showed that albumin production was regulated by cell density during the growth cycle. Albumin production in most cell lines was also regulated by dexamethasone; however, one cell line continued to produce high levels of albumin when the cells were grown in medium lacking dexamethasone, demonstrating that although glucocorticoid can induce albumin production in some cell lines, it is not required for high levels of albumin production by all cells in culture. Regulation of albumin production measured at the level of protein secretion was paralleled by changes in steady-state levels of a 2.3-kilobase albumin RNA. Albumin-producing SV40-immortalized hepatocytes secreted a variety of other plasma proteins, including transferrin, hemopexin, and the third component of complement. These cells also expressed tyrosine aminotransferase activity that was inducible by dexamethasone. Alpha-fetoprotein production was not detected in any of the cell lines examined.     

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

The nucleotide sequence of rabbit liver transferrin cDNA

The cDNA sequence of rabbit liver transferrin has been determined. The largest cDNA was 2279 base pairs (bp) in size and encoded 694 amino acids consisting of a putative 19 amino acid signal peptide and 675 amino acids of plasma transferrin. The deduced amino acid sequence of rabbit liver transferrin shares 78.5% identity with human liver transferrin and 69.1% and 44.8% identity with porcine and Xenopus transferrins, respectively. At the amino acid level, vertebrate transferrins share 26.4% identity and 56.5% similarity. The most conserved regions correspond to the iron ligands and the anion binding region. Optimal alignment of transferrin sequences required the insertion of a number of gaps in the region corresponding to the N-lobe. In addition, the N-lobes of transferrins share less amino acid sequence similarity than the C-lobes.     

Length polymorphisms at two candidate genes explain variation of migratory behaviors in blackpoll warblers (Setophaga striata)

Migratory behaviors such as the timing and duration of migration are genetically inherited and can be under strong natural selection, yet we still know very little about the specific genes or molecular pathways that control these behaviors. Studies in candidate genes Clock and Adcyap1 have revealed that both of these loci can be significantly correlated with migratory behaviors in birds, though observed relationships appear to vary across species. We investigated geographic genetic structure of Clock and Adcyap1 in four populations of blackpoll warblers (Setophaga striata), a Neotropical–Nearctic migrant that exhibits geographic variation in migratory timing and duration across its boreal breeding distribution. Further, we used data on migratory timing and duration, obtained from light‐level geolocator trackers to investigate candidate genotype–phenotype relationships at the individual level. While we found no geographic structure in either candidate gene, we did find evidence that candidate gene lengths are correlated with five of the six migratory traits. Maximum Clock allele length was significantly and negatively associated with spring arrival date. Minimum Adcyap1 allele length was significantly and negatively associated with spring departure date and positively associated with fall arrival date at the wintering grounds. Additionally, we found a significant interaction between Clock and Adcyap1 allele lengths on both spring and fall migratory duration. Adcyap1 heterozygotes also had significantly shorter migration duration in both spring and fall compared to homozygotes. Our results support the growing body of evidence that Clock and Adcyap1 allele lengths are correlated with migratory behaviors in birds.     

Establishment and optimization of epithelial cell cultures from human ectocervix,transformation zone,and endocervix optimization of epithelial cell cultures

Cervical cancer is a major public health problem and research using cell culture models has improved understanding of this disease. The human cervix contains three anatomic regions; ectocervix with stratified squamous epithelium, endocervix with secretory epithelium, and transformation zone (TZ) with metaplastic cells. Most cervical cancers originate within the TZ. However, little is known about the biology of TZ cells or why they are highly susceptible to carcinogenesis. The goal of this study was to develop and optimize methods to compare growth and differentiation of cells cultured from ectocervix, TZ or endocervix. We examined the effects of different serum-free media on cell attachment, cell growth and differentiation, and cell population doublings in monolayer culture. We also optimized conditions for organotypic culture of cervical epithelial cells using collagen rafts with human cervical stromal cells. Finally, we present a step-by-step protocol for culturing cells from each region of human cervix.     

Novel role for E4 region genes in protection of adenovirus vectors from lysis by cytotoxic T lymphocytes

Target cells infected with adenovirus (Ad) vectors containing intact E3 and E4 regions were found to be relatively resistant to lysis by Ad-specific cytotoxic T lymphocytes. Elements from both the E3 and the E4 regions were required for this effect, leading to the identification of a previously undescribed role for E4 gene products in resistance to cytolysis.