Growth, carcass quality, and protein and energy metabolism in beef cattle with different growth potentials and residual feed intakes.

Twenty-four beef steers (predominantly Angus x Hereford, 14 to 18 mo of age, 403 +/- 3 kg of BW), were housed and fed in individual pens for about 122 d. Twelve steers came from a herd that had been selected for growth (high growth; HG) and the other 12 from a herd with no selection program (low growth; LG). Another 6 steers (3 from each group) were slaughtered at the beginning to obtain the initial composition. All steers were fed the same corn-based diet (3.06 Mcal of ME/kg of DM, 13.6% CP) on an ad libitum basis. Two weeks before slaughter, total urine was collected for 5 d for estimation of 3-methylhistidine excretion and myofibrillar protein breakdown rates. Compared with LG steers, HG steers had less initial BW but greater final BW, DMI (7.52 vs. 6.37 kg/d), ADG (1.33 vs. 0.853 kg/d), G:F (0.176 vs. 0.133 kg/kg), ME intake (0.233 vs. 0.201 Mcal x kg of BW(0.75) x d(-1)), and retained energy (RE; 0.0711 vs. 0.0558 Mcal x kg of BW(0.75) x d(-1)); gained more fat (676 vs. 475 g/d); and tended to gain more whole body protein (100 vs. 72 g/d), with no difference in residual feed intake (RFI). Estimated net energetic efficiency of gain (k(g)) and ME for maintenance (ME(m)) did not differ between the 2 groups, averaging 0.62 and 0.114, respectively. The HG steers had greater HCW (350 vs. 329 kg), backfat (16.1 vs. 11.6 mm), and yield grades (3.53 vs. 2.80), with a similar dressing percent, KPH fat, LM area, and marbling score. Skeletal muscle protein gain (70.2 vs. 57.6 g/d) and fractional protein accretion rate (0.242 vs. 0.197%/d) tended to be greater in HG than in LG steers. Steers were classified into low (-0.367 kg/d) and high (0.380 kg/d) RFI classes. Compared with the high RFI steers, low RFI steers consumed less DM (6.61 vs. 7.52 kg/d) and ME (0.206 vs. 0.234 Mcal x kg of BW(0.75) x d(-1)) and tended to gain less fat (494 vs. 719 g/d), but were similar for initial and final BW, ADG, G:F, protein gain, HCW, dressing percent, backfat, KPH fat, LM area, marbling score, and yield grade, as well as for all observations related to myofibrillar protein metabolism. Residual feed intake may be positively [corrected] correlated with ME for maintenance. The maintenance energy requirement increased by 0.0166 Mcal x kg(-0.75) x d(-1) for each percentage increase in fractional protein degradation rate, confirming the importance of this process in the energy economy of the animal.

Goliath, a ring-H2 mitochondrial protein, regulated by luteinizing hormone/human chorionic gonadotropin in rat leydig cells.

We have cloned the rat homologue of the ring-H2 protein Goliath involved in Drosophila development. The rat Goliath mRNA (1.85 kb) was translated as a major ubiquitous protein species of 28-kDa and three larger isoforms (50, 46, and 36 kDa) expressed mainly in liver, lung, stomach, heart, and thymus and barely detectable in other tissues (kidney, skeletal muscle, brain, testis, intestine, and spleen). By immunohistochemistry on rat testis sections, we localized the protein in interstitial tissue and seminiferous tubules. In tubules, Goliath was expressed mainly in postmeiotic germ cells and to a much lesser extent in Sertoli cells. In the interstitium, Goliath was exclusively present in Leydig cells. Using a series of immunolabeling, cellular fractionation, and electron microscopy experiments, we established that Goliath is present in mitochondria of the R2C Leydig cell line. Using short-term hypophysectomized animals, we showed that Goliath is regulated by LH/hCG in Leydig cells but not in germ cells. This regulation in Leydig cells concerned only the 50-kDa isoform. This report is the first description of a differential regulation of the Goliath protein between germ cells and Leydig cells.

Sporadic testicular germ cell cancers do not exhibit specific alteration in CAG/CTG repeats containing genes expressed in human testis.

CAG/CTG repeat expansions in genomic DNA of testicular tumour cell lines, and germline DNA from members of families predisposed to this malignancy, have been previously described. In order to identify genes possibly concerned by this alteration, we attempted to clone all possible human testis cDNA containing at least five CAG/CTG repeats. Thirty-four different transcripts were identified. By using PCR and non denaturing gel electrophoresis, we determined the size of their repeats, as well as their polymorphisms in a collection of human testicular germ cell tumours and the normal surrounding tissues. For all tested genes, we detected the presence of several species of the same mRNA for each person. Nine genes exhibited specific patterns of expression among different groups of individuals, indicative of polymorphism. None of these polymorphisms was related to human testicular tumours.

hH-Rev107, a class II tumor suppressor gene, is expressed by post-meiotic testicular germ cells and CIS cells but not by human testicular germ cell tumors.

By systematic analysis of a human testis library, we have isolated the hH-Rev107-3 cDNA, identical to hH-Rev107-1 cDNA, which was previously described as a class II tumor suppressor gene. In this study, two transcripts (1 and 0.8 kb) were detected by Northern blot in all human tissues, excepted in thymus. The strongest expression was found in testis, skeletal muscle and heart. These two mRNA are probably transcribed from only one gene that we mapped to the q12-q13 region of the chromosome 11. In human testis, hH-Rev107 gene expression was localized, by in situ hybridization, within the round spermatids. To investigate a possible role for hH-Rev107 protein in testicular malignant growth, we examined the expression of this gene in germ cell tumors. A strong hH-Rev107 gene expression was observed in normal testis as well as in samples with preinvasive carcinoma in situ but was completely absent in overt tumors, both seminomas and non-seminomas. By in situ hybridization, CIS was found hH-Rev107 positive and tumor negative. A semi-quantitative assessment of hH-Rev107 mRNA level in testicular germ cell tumors, by RT-PCR, exhibited a ninefold decrease in the gene expression. No gross structural aberrations of hH-Rev107 gene were detected in these human primary tumors. The results suggest that down-regulation of hH-Rev107 may be associated with invasive progression of testicular germ cell tumors.

Identification and mapping of 26 human testis mRNAs containing CAG/CTG repeats.

Various types of pathologies, including neurodegenerative diseases, as well as different types of neoplasia, are related to genes exhibiting simple tandem repeat instabilities. In order to seek for new candidate genes for such disorders, we screened 4.10(6) human testis cDNAs for CAG- and CTG-containing clones. Among 910 positive clones, we characterized 109 cDNAs corresponding to 26 independent mRNAs. Fourteen of these mRNAs represent new genes. The corresponding clones contain between 3 and 19 consecutive CAG or CTG triplets. We assigned 15 out of these 26 genes to 14 different human chromosomes. These genes represent new potential candidates for diseases associated with CAG or CTG repeat mutations.

Identification and chromosomal localization of human genes containing CAG/CTG repeats expressed in testis and brain.

Human genes containing triplet repeats have been demonstrated to be involved in several neurodegenerative diseases by expansion of the repeat in succeeding generations. To identify novel genes involved in such pathologies, we have isolated transcripts containing (CAG/CTG)n repeats using two approaches. First, we screened 4 x 10(6) clones representing 10 copies of a human testis cDNA library using a (CAG)14 oligonucleotide probe. Among the 910 clones identified, the 243 clones with the strongest hybridization signal were sequenced partially from 3' or 5' ends. This provided us with 251 partial sequences that grouped into clusters corresponding to 39 genes, of which 19 represent unknown species. Second, we selected 203 additional ESTs containing (CAG/CTG)n repeats representing 121 clusters from the IMAGE consortium infant brain cDNA library. From these two series of sequences, we have localized 95 genes on human chromosomes using a panel of whole genome radiation hybrid (Genebridge 4). These genes are located on all of the chromosomes except for chromosome X, the highest density being observed on chromosome 19.

High-level expression of functional human gamma-glutamyl transpeptidase using the baculovirus system.

The understanding of the structure and function of gamma-glutamyl transpeptidase (GGT) has been hindered by the difficulty of obtaining large quantities of functional enzyme. A recombinant baculovirus, encoding the human hepatoma cell (Hep G2) GGT, was easily purified using a histochemical procedure to reveal GGT activity. Infected insect cells synthesized a large amount of enzymatically active GGT representing up to 10% of the total cell extract protein. The GGT specific activity of the infected cells was 13 units per mg of protein which is the highest GGT expression level reported to date, 260-times more than in Hep G2 cells. The recombinant protein displayed an apparent molecular mass (M(r), 58,000 for the heavy subunit), immunoreactivity and catalytic features similar to those of the native protein. The high-level expression of functional GGT should provide an excellent tool to further study the structure-function relationships of the protein.

Human testis specifically expresses a homologue of the rodent T lymphocytes RT6 mRNA.

A human homologue of the rodent T cell mono ADP-ribosyl transferase RT6 mRNA was identified by a systematic analysis of human testis transcripts. This messenger encodes for a precursor protein of 367 aa (MW: 41.5 kDa) which exhibits a peptide signal, consensus domains for mono ADP-ribosyl transferase and a C-terminal part characteristic of glycophosphatidyl inositol anchored protein. This mRNA, transcribed from a gene localized in 4q13-q21, is not expressed in white blood cells but is specific for human testis in which it is likely to correspond to a new ADP-ribosyl transferase.

Identification of a cat eye syndrome using DNA sequence dosage analysis.

We report here the detection of a Cat Eye Syndrome (CES) in a woman who does not exhibit the related phenotype, due to intensive surgery. The analysis of her karyotype reveals a small supernumerary bisatellited chromosome likely to correspond to a fragment of chromosome 13, 15, 21 or 22 on banding analysis. Southern blot of genomic DNA of this patient and her parents hybridized with probes specific of these chromosomes, revealed a DNA amplification of the 22q11 region for the patient, likely to correspond to a CES.

Characterization of a large population of mRNAs from human testis.

We present the results of single-pass sequencing of 779 expressed sequence tags from normal human testis cDNA clones. Of the sequences generated, 319 (41%) appeared to be completely unknown and are likely to represent new genes, and 289 (37%) were identified based on exact or nonexact matches to sequences in public databases. In analyses of hybridization of four tissues, testis, brain, liver, and kidney, 6 of 12 cDNAs clones revealed testis-specific expression. This argues for the value of the combination of random sequencing and analysis of cellular expression for large-scale characterization of gene expression in the testis.

Repetitive 5-azacytidine treatments of Fao cells induce a stable and strong expression of gamma-glutamyl transpeptidase.

The role of DNA methylation in the expression of the rat gamma-glutamyl transpeptidase (GGT) gene was assessed in the Fao cell line using a hypomethylating agent, 5-azacytidine. Ten repetitive treatments of the cells, with 8 microM 5-azacytidine for 24 h, led to 13- and 80-fold increases, respectively, in GGT activity and in GGT mRNA level. The DNA methylation patterns generated by the isoschizomeric restriction enzymes Hpa II and Msp I indicated that the GGT gene, highly methylated in Fao cells, became strongly demethylated after 5-azacytidine treatments. Thus, DNA demethylation increases the expression of the GGT gene. 5-Azacytidine treatments also increased, but to a lesser extent, mRNAs level for actin, albumin, mitochondrial aspartate aminotransferase, aldolase B mRNAs (12- to 16-fold) as well as for tubulin, gluthathione transferase, and tyrosine aminotransferase mRNAs (2- to 5-fold). The GGT gene expression was further studied in B4 cells, cloned from the demethylated Fao cell population. This clone B4 exhibited a stable and strong GGT activity and a highly demethylated GGT gene. Among the three GGT mRNA I, II, or III, transcribed from three different promoters of the single rat GGT gene, only mRNA III was detected in Fao cells and was increased in clone B4, indicating that the demethylation acts on the promoter for mRNA III. The analysis of the differentiation state of B4 cells, as compared to Fao cells, showed a loss of the regulation of GGT and aspartate aminotransferase genes by dexamethasone, as well as a loss of the gluconeogenic pathway. Interestingly, B4 cells have retained many other specific functions of hepatic differentiation and have acquired alpha-fetoprotein expression; thus this clone exhibits the characteristics of a hepatic fetal phenotype.

Tissue- and developmental-stage-specific methylation in the two kidney promoters of the rat gamma-glutamyl transpeptidase gene.

We have investigated, using DNA methylation patterning, the site-specific methylation of promoters I and II of the rat gamma-glutamyl transpeptidase gene. This analysis was done in fetal, newborn and adult rat kidney, in which promoters I and II are progressively active during development, as well as in rat liver, which never expresses mRNAs from these two promoters. During kidney development, a progressive demethylation occurs in the promoter I and II region, specially at the level of the most proximal MspI site of promoter II. A progressive reorganization of the methylated sites within the 5' end of the gene also occurs during liver development.

Molecular cloning of the cDNAs coding for the two subunits of soluble guanylyl cyclase from human brain.

Complementary DNA clones corresponding to the 70 and 82 kDa subunits of soluble guanylyl cyclase from human adult brain have been isolated and sequenced. Their respective open reading frames correspond to 619 amino acids (M(r) 70,469) and 717 amino acids (M(r) 81,324). Southern blots of human genomic DNA using these clones as probes give patterns which might be compatible with the presence of more than one copy per gene, or pseudogenes, for each subunit in the human genome. Comparison of the protein sequence of the large subunit from adult brain with the subunit cloned from human fetal brain (Harteneck, C., Wedel, B., Koesling, D., Malekewitz, J., Böhme, E., and Schultz, G. (1991) FEBS Lett. 292, 217-222) revealed only 34% homology. This result demonstrates the existence of a novel large subunit isoform for soluble guanylyl cyclase in human tissues.

Pattern of expression of gamma-glutamyl transpeptidase in rat liver and kidney during development: study by immunochemistry and in situ hybridization.

We used an in situ hybridization technique using single-stranded RNA probes to study the expression pattern of gamma-glutamyl transpeptidase (GGT) in rat liver and kidney during development. The results were compared to those obtained with an immunoperoxidase technique and with Northern blot analysis of GGT mRNA. In the kidney, northern blot revealed a 20-fold increase of GGT mRNA between day 18 of gestation and adulthood. Protein and mRNA localization clearly identified the proximal tubules as the site of synthesis of GGT. In the liver, the expression was lower than in the kidney and Northern blot showed a dramatic decrease of expression after birth. Using immunohistochemistry, the protein was detected within parenchymal cells in embryo and hepatocyte membranes and bile ducts in adults. Using in situ hybridization, GGT mRNA was only detected on days 1 and 2 after birth and exclusively in hepatocytes. Immunoperoxidase may be more sensitive than in situ hybridization to study the expression of minor liver protein such as GGT. However, the study of GGT expression using in situ hybridization is possible in cases of increased expression such as alcoholism, cholestasis, and carcinogenesis.

Localization by in situ hybridization of gamma-glutamyl transpeptidase mRNA in the rat kidney using 35S-labeled RNA probes.

gamma-Glutamyl transpeptidase (GGT) is involved in the extraction of plasma glutathione in the postglomerular compartment of the kidney. The enzyme is distributed in the proximal tubule associated with the apical brush border. Using in situ hybridization with 35S-labeled anti-sense and sense RNA GGT probes on rat kidney cryostat sections, the present study demonstrates that GGT mRNA transcripts are detected in proximal tubules localized in the inner cortex, outer medulla, and medullary rays of the cortex. Such a distribution suggests that the GGT gene is mainly expressed in the more distal part of the proximal tubule, i.e., the pars recta. As a control, in situ hybridization has also been performed using a 35S-labeled beta-actin anti-sense RNA probe showing a diffuse pattern of distribution of beta-actin RNA transcripts particularly in the outer cortex. This highly sensitive method using single strand asymetric RNA probes, with a markedly reduced nonspecific binding is promising for the study of gene expression patterns in the kidney in order to clarify their heterogeneity in the various segments of the nephron.

Regulation of mouse mammary-gland gamma-glutamyltranspeptidase mRNA during pregnancy, lactation and weaning.

The level of gamma-glutamyltranspeptidase (GGT) activity and of its mRNA were determined in the mouse mammary gland during pregnancy, lactation and weaning. The GGT activity, which is very low in the virgin-mouse mammary gland (5 munits/mg of protein), increases progressively during pregnancy (3-fold), reaches its maximum at the onset of lactation (8-fold) and returns rapidly to basal level at weaning. Although no GGT-specific mRNA is detected in the virgin-mouse mammary gland, a single faint band of 2.2 kb in size is found during pregnancy. During lactation, an additional mRNA of 2.4 kb in size appears, and the level of both mRNAs is higher. This high level of mRNA persists during weaning as well. Southern-blot analysis of mouse mammary-gland DNA provides convincing evidence that there is only one gene which codes for the two mRNAs. The present study provides the first evidence for a physiological regulation of the two GGT mRNAs in the same tissue.

Mechanism of gamma-glutamyl transpeptidase release in serum during intrahepatic and extrahepatic cholestasis in the rat: a histochemical, biochemical and molecular approach.

The mechanism of the elevation of serum gamma-glutamyl transpeptidase activity in cholestasis is not clear. We therefore analyzed rat gamma-glutamyl transpeptidase activities in liver, bile and serum during intrahepatic cholestasis induced by a single dose of alpha-naphthyl isothiocyanate (20 mg/100 gm body weight) and during extrahepatic cholestasis after bile duct ligation. At days 1 and 2 after alpha-naphthyl isothiocyanate ingestion, we saw a fivefold and a 60-fold increase in serum and bile gamma-glutamyl transpeptidase activities, respectively. These increases were associated with a decrease in hepatic gamma-glutamyl transpeptidase activity and of corresponding mRNA. Simultaneously, necrosis of the biliary epithelium appeared in portal tracts. From day 2 to day 14, gamma-glutamyl transpeptidase activity in bile and serum progressively returned to basal levels; in the liver, cholangiolar proliferation was mild and was associated with moderate elevation of the gamma-glutamyl transpeptidase activity and of its corresponding mRNA. In extrahepatic cholestasis, a 10-fold increase in serum gamma-glutamyl transpeptidase activity was detected between day 0 and day 14. This increase was associated with major cholangiolar proliferation and with a progressive rise in hepatic gamma-glutamyl transpeptidase activity and in specific mRNA; in bile, gamma-glutamyl transpeptidase activity was slightly elevated. In these two models of cholestasis, histochemically detected gamma-glutamyl transpeptidase activity was largely predominant in biliary cells. We found no significant induction of gamma-glutamyl transpeptidase activity in hepatocytes. These results suggest that in these two models of cholestasis, the increase in serum gamma-glutamyl transpeptidase activity is of biliary cell origin and does not originate from hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

An alternatively processed mRNA specific for gamma-glutamyl transpeptidase in human tissues.

Human gamma-glutamyl transpeptidase (GGT)1 is composed of two subunits derived from a single precursor (Nash, B., and Tate, S.S. (1984) J. Biol. Chem. 259, 678-685; Finidori, J., Laperche, Y., Tsapis, R., Barouki, R., Guellaën, G., and Hanoune, J. (1984) J. Biol. Chem. 259, 4687-4690) consisting of 569 amino acids (Laperche, Y., Bulle, F., Aissani, T., Chobert, M.N., Aggerbeck, M., Hanoune, J., and Guellaën, G. (1986) Proc Natl. Acad. Sci. U.S.A. 83, 937-941). In the present study we report the cloning of an altered form of this precursor from human liver. We have isolated two clones, one 2,632 base pairs (bp) long from a fetal liver cDNA library and one 926 bp long from an adult liver cDNA library, each containing a 22-bp insertion that introduces a premature stop codon and shortens the open reading frame to 1,098 bp when compared with known human cDNA sequences specific for GGT. Sequence analysis of a human genomic GGT clone shows that this insertion of 22 bp is generated by a splicing event involving an alternative 3'-acceptor site. By polymerase chain reaction experiments we demonstrate that the alternatively spliced mRNA is present in polysomes from the microsomal fraction of a human hepatoma cell line (Hep G2) and thus could encode an altered GGT molecule of 39,300 Da (366 amino acids) encompassing most of the heavy subunit which is normally 41,500 Da (380 amino acids). The altered mRNA is detected in various human tissues including liver, kidney, brain, intestine, stomach, placenta, and mammary gland. This report is the first demonstration of an alternative primary sequence in the mRNA coding for GGT, a finding that could be related to the presence of some inactive forms of GGT detected in human tissues.

Different gamma-glutamyl transpeptidase mRNAs are expressed in human liver and kidney.

In human, the two subunits of gamma-glutamyl transpeptidase (GGT) arise from a common precursor encoded by a multigene family. Until now, a single specific coding sequence for this precursor (type I) has been identified in human placenta and liver. In the present study, we have isolated from a human kidney cDNA library, a GGT specific clone (0.8 Kb). The sequence of which (type II) i) covers the carboxy terminal part of the GGT precursor, ii) exhibits 22 point mutations and a 30 bp deletion as compared to the type I GGT sequence. The sequencing of a human genomic clone reveals that this type II GGT mRNA is encoded by a different gene than the type I GGT mRNA. Both type I and type II GGT mRNAs are expressed in human liver, while almost exclusively type II GGT mRNA is detected in human kidney.

Chromosomal localization of human aspartate aminotransferase genes by in situ hybridization.

The localization of the human genes for cytosolic and mitochondrial aspartate aminotransferase (AspAT) has been determined by chromosomal in situ hybridization with specific human cDNA probes previously characterized in our laboratory. The cytosolic AspAT gene is localized on chromosome 10 at the interface of bands q241-q251. Mitochondrial AspAT is characterized by a multigene family located on chromosomes 12 (p131-p132), 16 (q21), and 1 (p32-p33 and q25-q31). Genomic DNA from ten blood donors was digested by ten restriction enzymes, and Southern blots were hybridized with the two specific probes. Restriction fragment length polymorphism was revealed in only one case for cytosolic AspAT, with PvuII, while no polymorphism for mitochondrial AspAT was found.