Tyrosine aminotransferase induced in cells genetically and epigenetically deficient for this enzyme

Mice homozygous for lethal deletions around the albino (c) locus are deficient for several liver-specific enzymes, including tyrosine aminotransferase (TAT). The structural gene coding for this enzyme appears to be intact in these mutants and can be “activated” in homozygous mutant mouse liver-rat hepatoma cell hybrids. The present study demonstrates that the mouse form of TAT can also be induced in both normal and mutant mouse skin-rat hepatoma cell hybrids. Thus, a liver-specific enzyme is expressed in skin cells, both normal and mutant, the normal differentiated state of which excludes TAT expression.     

Isolation and characterization of the human tyrosine aminotransferase gene.

Structure and sequence of the human gene for tyrosine aminotransferase (TAT) was determined by analysis of cDNA and genomic clones. The gene extends over 10.9 kbl and consists of 12 exons giving rise to a 2,754 nucleotide long mRNA (excluding the poly(A)tail). The human TAT gene is predicted to code for a 454 amino acid protein of molecular weight 50,399 dalton. The overall sequence identity within the coding region of the human and the previously characterized rat TAT genes is 87% at the nucleotide and 92% at the protein level. A minor human TAT mRNA results from the use of an alternative polyadenylation signal in the 3' exon which is present but not used at the corresponding position in the rat TAT gene. The non-coding region of the 3' exon contains a complete Alu element which is absent in the rat TAT gene but present in apes and old world monkeys. Two functional glucocorticoid response elements (GREs) reside 2.5 kb upstream of the rat TAT gene. The DNA sequence of the corresponding region of the human TAT gene shows the distal GRE mutated and the proximal GRE replaced by Alu elements.     

Establishment and partial characterization of SV40 virus-immortalized hepatocyte lines of normal and lethal mutant mice carrying a deletion on chromosome 7

Deletions in chromosome 7 of the mouse have been shown to cause failure of expression of various hepatocyte-specific genes in newborn deletion homozygotes, including the gene encoding tyrosine amino transferase (TAT) (EC 2.6.1.5) (Gluecksohn-Waelsch, 1979). Primary liver cultures of newborn albino deletion mutant mice (c14CoS/c14CoS) and of phenotypically normal mice (c14CoS/cch or cch/cch) were infected with SV40 virus and multiplying hepatocytes selected in arginine-deficient medium containing epidermal growth factor (EGF), insulin, and hydrocortisone (HC). Resulting normal (NMH-ch) and mutant (NMH-m14) hepatocyte lines expressing integrated viral transforming sequences did not senesce, they multiplied autonomously of EGF in medium with insulin plus HC, and they retained hepatocyte-specific functions. Both lines synthesized arginine and contained albumin and alpha-fetoprotein (AFP) mRNAs. TAT-specific mRNA was detected in normal but not in mutant hepatocyte lines. A fragment of the mouse tyrosinase gene, known to map at the albino locus (c) within the region deleted in the c14CoS mutant, hybridized with a 2.5 kb EcoRI fragment of normal NMH-ch DNA, whereas this fragment was undetectable in mutant NMH-m14 DNA. These immortalized hepatocyte lines reflect important properties of normal and mutant liver tissues from which they were derived. The deletion mutant mouse cell lines may be useful for complementation studies involving sequences corresponding to the deletions that encode regulatory gene(s) involved in the control of inducible expression of certain hepatocyte-specific genes such as TAT.     

cDNA sequence and chromosomal localization of the mouse parvalbumin gene, Pva

In the homozygous condition, the mutation adr (arrested development of righting response) of the mouse causes a myotonia and a drastic reduction of the Ca2+-binding protein parvalbumin (PV) in fast muscles. Using a rat PV probe, a mouse cDNA clone was isolated from a lambda gt11 wild-type fast-muscle library and its nucleotide sequence was determined. The protein coding and the 3' nontranslated regions of the mouse gene show extensive homology with the rat PV gene. The result of Southern blot hybridization is consistent with a single copy gene for parvalbumin. Restriction fragment length polymorphisms (RFLPs) between Mus musculus domesticus (e.g. C57BL/6) and Mus spretus (SPE) were detected with the enzymes Eco RI, Pst I, and Sst I. The restriction fragment patterns of DNA samples from 65 individual offspring of (C57BL/6 x SPE)F1 x C57BL/6 backcrosses were tested with the PV probe and matched, for linkage detection, to pre-existing patterns established with various RFLP probes on the same samples. A co-distribution of PV-RFLPs with Pvt-1 and Mlvi-2, which had been localized on chromosome 15, was detected. Thus, the structural gene for PV, designated Pva, maps to chromosome 15 of the mouse whereas the adr mutation shows no linkage with markers on this chromosome. Gene locus homology between chromosome 15 of the mouse and chromosome 22 of man (which carries the human PV gene) is discussed.     

Characterization of the cDNA coding for mouse prothrombin and localization of the gene on mouse chromosome 2

A series of overlapping cDNAs coding for mouse prothrombin (coagulation factor II) have been isolated and the composite DNA sequence has been determined. The complete prothrombin cDNA is 1,987 bp in length [excluding the poly(A) tail] and codes for 18 bp of 5' untranslated sequence, an open reading frame coding for 618 amino acids, a stop codon, and a 3' untranslated region of 112 bp followed by a poly(A) tail. The translated amino acid sequence predicts a molecular weight of 66,087, which includes 10 residues of gamma-carboxyglutamic acid. There are five potential N-linked glycosylation sites. Mouse prothrombin is 81.4% and 77.3% identical to the human and bovine proteins, respectively. Comparison of the cDNA coding for mouse prothrombin to the human and bovine cDNAs indicates 79.9% and 76.5% identity, respectively. Amino acid residues important for the structure and function of human prothrombin are conserved in the mouse and bovine proteins. In the adult mouse and rat, prothrombin is primarily synthesized in the liver, where is constitutes 0.07% of total mRNA as determined by solution hybridization analysis. The genetic locus for mouse prothrombin, Cf-2, has been mapped using an interspecies backcross and DNA fragment differences between the two species. The prothrombin locus lies on mouse chromosome 2, 1.8 +/- 1.3 map units proximal to the catalase locus. The gene order in this region is Cen-Acra-Cf-2-Cas-1-A-Tel. This localization extends the proximal boundary of the known region of homology between mouse chromosome 2 and human chromosome 11p from Cas-1 about 2 map units toward the centromere.     

Structure and mapping of the gene encoding mouse high affinity Fc gamma RI and chromosomal location of the human Fc gamma RI gene.

We describe the isolation and characterization of the gene encoding the mouse high affinity Fc receptor Fc gamma RI. Using a mouse cDNA Fc gamma RI probe four unique overlapping genomic clones were isolated and were found to encode the entire 9 kb of the mouse Fc gamma RI gene. Sequence analysis of the gene showed that six exons account for the entire Fc gamma RI cDNA sequences including the 5'- and 3'-untranslated sequences. The first and second exons encode the signal peptide; exons 3, 4, and 5 encode the extracellular Ig binding domains; and exon 6 encodes the transmembrane domain, the cytoplasmic region, and the entire 3'-untranslated sequence. This exon pattern is similar to Fc gamma RIII and Fc epsilon RI but differs from the related Fc gamma RII gene which contains 10 exons and encodes the b1 and b2 Fc gamma RII. Southern blot analysis had shown that the mouse Fc gamma RI gene is a single copy gene with no RFLP in inbred strains of mice, but analysis of an intersubspecies backcross of mice showed that unlike other mouse FcR genes which are on mouse chromosome 1 the locus encoding Fc gamma RI, termed Fcg1, is located on chromosome 3. Interestingly, the Fcg1 locus is located near the end of a region with known linkage homology to human chromosome 1. Analysis of human x rodent somatic cell hybrid cell lines indicates that the human FCG1 locus encoding the human Fc gamma RI maps to chromosome I and therefore possibly linked to other FcR genes on this chromosome. These results suggest that the linkage relationships among these genes in the human genome are not preserved in the mouse.     

Chromosomal assignment and trans regulation of the tyrosine aminotransferase structural gene in hepatoma hybrid cells.

The structural gene encoding liver-specific tyrosine aminotransferase (TAT; EC 2.6.1.5) was assigned to mouse chromosome 8 by screening a series of hybrid cell lines for retention of murine Tat-1 gene sequences by genomic Southern blotting. This assignment demonstrated that the Tat-1 structural gene was not syntenic with Tse-1, a chromosome 11-linked locus that negatively regulates TAT expression in trans (A. M. Killary and R. E. K. Fournier, Cell 38:523-534, 1984). We also showed that the fibroblast Tat-1 gene was systematically activated in hepatoma X fibroblast hybrids retaining fibroblast chromosomes 8 in the absence of chromosome 11 but was extinguished in cells retaining both fibroblast chromosomes. Thus, the TAT structural genes of both parental cell types were coordinately regulated in the intertypic hybrids, and the TAT phenotype of the cells was determined by the presence or absence of fibroblast Tse-1.     

Assignment of the human tyrosine aminotransferase gene to chromosome 16

Summary The liver enzyme tyrosine aminotransferase (TAT; EC 2.6.1.5) catalyzes the rate-limiting step in the catabolic pathway of tyrosine. Deficiency in TAT enzyme activity underlies the autosomally inherited disorder tyrosinemia II (Richner-Hanhart syndrome). Using a human TAT cDNA clone as hybridization probe, we have determined the chromosomal location of the TAT structural gene by Southern blot analysis of DNAs from a series of human x rodent somatic cell hybrids. The results assign the TAT gene to human chromosome 16.     

Modulation of the agonist activity of antisteroids by a novel cis-acting element.

The amount of agonist activity displayed by the antiglucocorticoid dexamethasone mesylate (Dex-Mes) for the induction of tyrosine aminotransferase (TAT) in rat hepatoma cells is greater than for glutamine synthetase and varies over a period of weeks. This variation, which has been reproduced over a period of 40 h by changing the density of the cells, suggests the involvement of a trans-acting factor. The target of this proposed trans-acting factor has now been localized to the region between -3.9 to -2.9 of the rat TAT gene from experiments with cells that were stably transfected with hybrid TAT/CAT constructs. Deletion experiments with transiently transfected TAT/tk promoter/CAT constructs revealed that this entire activity could be conveyed by a 21 bp sequence of the TAT gene. Gel shift experiments support the binding of a factor(s) to this 21 bp sequence. Thus the activity of the antagonist Dex-Mes is relatively independent of steroid structure and is largely determined by the further interactions of a trans-acting factor with the cis-acting sequence. We call this novel sequence a glucocorticoid modulatory element. A model is advanced which accounts for almost all of the results concerning TAT induction by glucocorticoids. This same model may also be useful in explaining why the amount of agonist activity of most antisteroids varies, even for different genes within the same cell.     

A genetic analysis of extinction: trans-dominant loci regulate expression of liver-specific traits in hepatoma hybrid cells

Extinction is an operational term that refers to the lack of expression of tissue-specific traits that is generally observed in hybrid cells formed by fusing dissimilar cell types. To define the genetic basis of this phenomenon, a series of rat hepatoma x mouse fibroblast hybrids has been isolated and characterized. We report here that the extinction of hepatic marker traits in these clones was strictly correlated with the retention of five particular fibroblast chromosomes (autosomes 8, 9, 10, 11, and 13). In order to dissect this correlation into its component parts, hepatoma microcell hybrids containing single, specific fibroblast chromosomes were constructed. Hepatoma clones retaining only fibroblast chromosome 11 were specifically extinguished for liver-specific tyrosine aminotransferase (TAT) expression, while expression of four other hepatic traits and of numerous constitutive markers was unaffected. Furthermore, removal of fibroblast chromosome 11 from the populations by back-selection resulted in reexpression of TAT activity to full parental levels. These data define and localize a genetic locus, tissue-specific extinguisher-1 (Tse-1), which regulates hepatic TAT expression in trans. We also provide evidence that human Tse-1 resides on the homologous chromosome (human chromosome 17), and that hybrids retaining active Tse-1 loci lack TAT-specific mRNA.     

Comparison of the tyrosine aminotransferase cDNA and genomic DNA sequences of normal mink and mink affected with tyrosinemia type II

Type II tyrosinemia, designated Richner-Hanhart syndrome in humans, is a hereditary metabolic disorder with autosomal recessive inheritance characterized by a deficiency of tyrosine aminotransferase activity. Mutations occur in the human tyrosine aminotransferase gene, resulting in high levels of tyrosine and disease. Type II tyrosinemia occurs in mink, and our hypothesis was that it would also be associated with mutation(s) in the tyrosine aminotransferase gene. Therefore, the transcribed cDNA and the genomic tyrosine aminotransferase gene were sequenced from normal and affected mink. The gene extended over 11.9 kb and had 12 exons coding for a predicted 454-amino-acid protein with 93% homology with human tyrosine aminotransferase. FISH analysis mapped the gene to chromosome 8 using the Mandahl and Fredga (1975) nomenclature and chromosome 5 using the Christensen et al. (1996) nomenclature. The hypothesis was rejected because sequence analysis disclosed no mutations in either cDNA or introns that were associated with affected mink. This suggests that an unlinked gene regulatory mutation may be the cause of tyrosinemia in mink.     

Structural analysis of the locus containing the human C-reactive protein gene and its related pseudogene

The gene for human C-reactive protein (CRP) is mapped within a 34-kilobase pair genomic DNA segment identified by chromosome walking through overlapping DNA fragments cloned into a lambda phage library. Within 16 kilobase pairs upstream and downstream of the locus for the authentic CRP gene, only one other sequence homologous to that for CRP could be found. Sequencing analysis indicates this sequence to be a pseudogene with 50-80% region-specific homology. Comparison of the authentic CRP gene cloned from genomic DNA libraries independently prepared from three patients indicates no difference in the 5' and 3' flanking region, promoter region, or coding sequence. Only a polymorphism in the length of the poly(GT) stretch located in the intron is observed. There appears to be only one gene locus and copy per haploid chromosome for the authentic CRP gene and its pseudogene.     

Gene for lymphoid enhancer-binding factor 1 (LEF1) mapped to human chromosome 4 (q23-q25) and mouse chromosome 3 near Egf.

LEF-1 is a 54-kDa nuclear protein that is expressed specifically in pre-B and T-cells. It binds to a functionally important site in the T-cell receptor alpha enhancer and contributes to maximal enhancer activity. LEF-1 is a member of a family of regulatory proteins that share homology with the high mobility group protein 1 (HMG1). The location of the LEF1 gene on human and mouse chromosomes was determined by Southern blot analysis of DNA from panels of interspecies somatic cell hybrids using a murine cDNA probe. Human-specific DNA fragments were detected in all somatic cell hybrids that retained the human chromosomal region 4cen-q31.2. Fluorescent in situ hybridization with two biotin-labeled overlapping human genomic cosmids revealed a specific hybridization signal at 4q23-q25. The homologous locus in the mouse was mapped to chromosome 3 by Southern analysis of rodent x mouse hybrid cell DNA. This chromosomal location was confirmed by the use of a restriction fragment length polymorphism (RFLP) in recombinant inbred mouse strains. The results of this RFLP analysis indicated that the mouse Lef-1 gene was closely linked to Pmv-39 and Egf and was likely placed between these loci, both of which were previously mapped to distal mouse chromosome 3. Our mapping results did not suggest involvement of this gene in previously mapped genetic disorders or in known neoplasia-associated translocation breakpoints.     

Molecular cloning, chromosomal assignment, and nucleotide sequence of the feline homeobox HOX3A.

The feline homolog to the mammalian homeobox locus, HOX3A, was isolated by screening a domestic cat genomic library with the murine Hox-3.1 probe. The nucleotide sequence similarity of the feline homeobox was 96% to human HOX3A, 94% to mouse Hox-3.1, and 94% to rat R4. The deduced amino acid sequence (homeodomain) of this feline homeobox was identical to all homeodomains of these cognate genes. Using a panel of feline x rodent somatic cell hybrids, the HOX3A locus was assigned to feline chromosome B4. Human HOX3A and mouse Hox-3.1 have been mapped previously to human chromosome 12 and mouse chromosome 15, respectively, both of which share syntenic homology to feline chromosome B4. These data demonstrate evolutionary conservation of both HOX3A gene sequences and chromosomal location during mammalian evolution.