Structure, chromosome location, and expression of the human gamma-actin gene: differential evolution, location, and expression of the cytoskeletal beta- and gamma-actin genes.

The accumulation of the cytoskeletal beta- and gamma-actin mRNAs was determined in a variety of mouse tissues and organs. The beta-isoform is always expressed in excess of the gamma-isoform. However, the molar ratio of beta- to gamma-actin mRNA varies from 1.7 in kidney and testis to 12 in sarcomeric muscle to 114 in liver. We conclude that, whereas the cytoskeletal beta- and gamma-actins are truly coexpressed, their mRNA levels are subject to differential regulation between different cell types. The human gamma-actin gene has been cloned and sequenced, and its chromosome location has been determined. The gene is located on human chromosome 17, unlike beta-actin which is on chromosome 7. Thus, if these genes are also unlinked in the mouse, the coexpression of the beta- and gamma-actin genes in rodent tissues cannot be determined by gene linkage. Comparison of the human beta- and gamma-actin genes reveals that noncoding sequences in the 5'-flanking region and in intron III have been conserved since the duplication that gave rise to these two genes. In contrast, there are sequences in intron III and the 3'-untranslated region which are not present in the beta-actin gene but are conserved between the human gamma-actin and the Xenopus borealis type 1 actin genes. Such conserved noncoding sequences may contribute to the coexpression of beta- and gamma-actin or to the unique regulation and function of the gamma-actin gene. Finally, we demonstrate that the human gamma-actin gene is expressed after introduction into mouse L cells and C2 myoblasts and that, upon fusion of C2 cells to form myotubes, the human gamma-actin gene is appropriately regulated.     

Use of an androgenized mare as an aid in detection of estrus in mares

A study was conducted over a 2-mo period to compare estrus detection results obtained using an androgenized teaser mare with those obtained with a stallion, using the same group of 10 normally cyclic mares. The teaser mare was androgenized by administration of boldenone undecylenate (500 mg i.m. every 1 to 2 wk), and allowed to run loose with the mare group. Estrus was determined by observation of the group for a 30-min period daily. In the second month of the experiment, a marking harness was used on the androgenized mare to help detect mares mounted when in estrus. Estrous periods detected by each teasing method were 1) first month: stallion, 18; androgenized mare, 5; 2) second month: stallion, 16; androgenized mare, 9. There were no estrous periods detected by the androgenized mare that were not also detected by the stallion. Under these conditions, the androgenized mare was not an adequate estrus detection aid. Also discussed are the successful results of an independent trial on a breeding farm using an androgenized mare as an estrus detection aid.     

pH Dependence of Histidine Affinity for Blood-Brain Barrier Carrier Transport Systems for Neutral and Cationic Amino Acids

The effects of pH (3.5-7.5) on the brain uptake of histidine by the blood-brain barrier (BBB) carriers for neutral and cationic amino acids were tested, in competition with unlabeled histidine, arginine, or phenylalanine, with the single-pass carotid injection technique. Cationic amino acid ( [14C]arginine) uptake was increasingly inhibited by unlabeled histidine as the pH of the injection solution decreased. In contrast, the inhibitory effect of unlabeled histidine on neutral amino acid ( [14C]phenylalanine) uptake decreased with decreasing pH. Brain uptake indices with varying histidine concentrations indicated that the neutral form of histidine inhibited phenylalanine uptake whereas the cationic form competed with arginine uptake. Since phenylalanine decreased [14C]histidine uptake at all pH values whereas arginine did not, it was concluded that the cationic form of histidine had an affinity for the cationic carrier, but was not transported by it. We propose that the saturable entry of histidine into brain is, under normal physiological circumstances, mediated solely by the carrier for neutral amino acids.     

Cloning of human lysyl hydroxylase: complete cDNA-derived amino acid sequence and assignment of the gene (PLOD) to chromosome 1p36.3----p36.2.

Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 dimer, catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in peptide linkages. A deficiency in this enzyme activity is known to exist in patients with the type VI variant of the Ehlers-Danlos syndrome, but no amino acid sequence data have been available for the wildtype or mutated human enzyme from any source. We report the isolation and characterization of cDNA clones for lysyl hydroxylase from a human placenta lambda gt11 cDNA library. The cDNA clones cover almost all of the 3.2-kb mRNA, including all the coding sequences. These clones encode a polypeptide of 709 amino acid residues and a signal peptide of 18 amino acids. The human coding sequences are 72% identical to the recently reported chick sequences at the nucleotide level and 76% identical at the amino acid level. The C-terminal region is especially well conserved, a 139-amino-acid region, residues 588-727 (C-terminus), being 94% identical between the two species and a 76-amino-acid region, residues 639-715, 99% identical. These comparisons, together with other recent data, suggest that lysyl hydroxylase may contain functionally significant sequences especially in its C-terminal region. The human lysyl hydroxylase gene (PLOD) was mapped to chromosome 1 by Southern blot analysis of human-mouse somatic cell hybrids, to the 1p34----1pter region by using cell hybrids that contain various translocations of human chromosome 1, and by in situ hybridization to 1p36.2----1p36.3. This gene is thus not physically linked to those for the alpha and beta subunits of prolyl 4-hydroxylase, which are located on chromosomes 10 and 17, respectively.     

Human collagen gene COL5A1 maps to the q34.2----q34.3 region of chromosome 9, near the locus for nail-patella syndrome.

Type V collagen is a fibrillar collagen that is widely distributed in tissues as a minor component of extracellular matrix and is usually composed of one pro alpha 2 (V) and two pro alpha 1 (V) chains. In this report, recently isolated cDNA and genomic clones, which encode the pro alpha 1 (V) chain, are used as probes for hybridization to filter-bound DNA from a panel of human-mouse hybrid cell lines and for in situ hybridization to metaphase chromosomes. These studies establish the chromosomal location of the COL5A1 gene, which encodes the pro alpha 1 (V) chain, within segment 9q34.2----q34.3. These findings add to the previously characterized dispersion of collagen genes in the human genome, as this is the first example of a collagen locus on chromosome 9. In addition, these studies place COL5A1 near the locus for the genetic disorder, nail-patella syndrome (hereditary osteo-onychodysplasia), which also maps to 9q34.     

The human cationic amino acid transporter (ATRC1): physical and genetic mapping to 13q12-q14.

The product of the mouse Rec-1 locus is an integral membrane protein that determines susceptibility to infection by murine ecotropic retroviruses. Recently it has been determined that its role in normal cell metabolism is transport of the cationic amino acids, arginine, lysine, and ornithine across the plasma membrane. Southern blot analysis of genomic DNA from a panel of 48 mouse-human somatic cell hybrids assigned the human version of this gene, ATRC1, to chromosome 13. Chromosomal in situ hybridization localized the gene to 13q12-q14. A restriction fragment length polymorphism (RFLP) was detected with TaqI. There were two alleles with frequencies of 0.29 and 0.71. Pairwise linkage analysis established linkage between ATRC1 and ATP1AL1, D13S1, D13S6, D13S10, D13S11, D13S21, D13S22, D13S33, D13S36, and D13S37. Multilocus linkage analysis of five of the loci indicated that the most likely order of loci in this region was D13S11-ATP1AL1-ATRC1-D13S6-D13S33.     

Structural requirements for high affinity ligand binding by estrogen receptors: a comparative analysis of truncated and full length estrogen receptors expressed in bacteria, yeast, and mammalian cells.

In order to better understand the structural requirements for effective high affinity binding of estrogens and antiestrogens by the human estrogen receptor (ER), a comparative study was undertaken in which we examined: 1) native ER from the MCF-7 ER-positive human breast cancer cell line; 2) full length ER expressed in yeast; 3) the ER hormone binding domain (amino acid residues 302-595) expressed in yeast; 4) a bacterially expressed protein A fusion product encoding a truncated ER (amino acid residues 240-595); and 5) a synthetic peptide encompassing amino acids 510-551 of the ER. The binding parameters studied included affinity, kinetics, structural specificity for ligands, and stability. Full length ER expressed in yeast was very similar to the MCF-7 ER in its affinity [dissociation constant (Kd), 0.35 +/- 0.05 nM], dissociation rate (t1/2, 3-4 h at 25 C), and structural specificity for both reversible and covalently attaching affinity ligands. While the truncated ER expressed in yeast was similar to MCF-7 ER in its specificity of ligand binding, it showed a slightly reduced affinity for estradiol (Kd, 1.00 +/- 0.17 nM). The bacterially expressed ER also had a lower affinity for estradiol (Kd, 1.49 +/- 0.16 nM), which may be due in part to an increase in the dissociation rate (t1/2, 0.5 h at 25 C). The attachment of covalent affinity ligands and structural specificity for a variety of reversible ligands was comparable in the bacterially expressed ER to that observed for the receptors expressed in MCF-7 cells and yeast.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of the human gene allowing infection by gibbon ape leukemia virus to human chromosome region 2q11-q14 and to the homologous region on mouse chromosome 2.

Retrovirus receptors remain a largely unexplored group of proteins. Of the receptors which allow infection of human and murine cells by various retroviruses, only three have been identified at the molecular level. These receptors include CD4 for human immunodeficiency virus, Rec-1 for murine ecotropic virus, and GLVR1 for gibbon ape leukemia virus. These three proteins show no homology to one another at the DNA or protein level. Therefore, work to date has not shown any general relationship or structural theme shared by retroviral receptors. Genes for two of these receptors (CD4 and Rec-1) and several others which have not yet been cloned have been localized to specific chromosomes. In order to assess the relationship between GLVR1 and other retroviral receptors, we mapped the chromosome location of GLVR1 in human and mouse. GLVR1 was found to map to human chromosome 2q11-q14 by in situ hybridization and somatic-cell hybrid analysis. This location is distinct from those known for receptors for retroviruses infecting human cells. Glvr-1 was then mapped in the mouse by interspecies backcrosses and found to map to chromosome 2 in a region of linkage conservation with human chromosome 2. This mouse chromosome carries Rec-2, the likely receptor for M813, a retrovirus derived from a feral Asian mouse. These data raise the interesting possibility that Rec-2 and Glvr-1 are structurally related.