GH1 cells are a clonal strain of rat pituitary tumor cells which synthesize GH and PRL. We have previously demonstrated that these cells respond to physiological concentrations of L-T3 and L-T4 when cultured with medium supplemented with thyroidectomized calf serum to achieve a thyroid hormone-depleted state under cell culture conditions. In this study, we describe a method to deplete euthyroid calf serum of L-T3 and L-T4 using an anion exchange resin. We demonstrate that the procedure only minimally alters the low molecular weight anion components of the serum and does not change the total protein content or the electrophoretic pattern of serum proteins. Moreover, we show that euthyroid calf serum depleted of L-T3 and L-T4 by this procedure yields serum which, when used as a medium supplement, results in biological responses identical to those obtained with media supplemented with thyroidectomized calf serum. In addition, resin treatment does not alter the growth-promoting properties of the serum if the thyroid hormone concentration is restored. This procedure should be useful in preparing thyroid hormone-depleted serum for cell culture studies in situations where thyroidectomy is not feasible or would require surgical procedures on a large number of small animals.

Evidence is accumulating that aging is hormonally regulated by an evolutionarily conserved insulin/IGF-1 signalling (IIS) pathway. Mutations in IIS components affect lifespan in Caenorhabditis elegans, Drosophila melanogaster and mice. Most long-lived IIS mutants also show increased resistance to oxidative stress. In D. melanogaster and mice, the long-lived phenotype of several IIS mutants is restricted to females. Here, we analysed the relationship between IIS signalling, body height and longevity in humans in a prospective follow-up study. Based on the expected effects (increased or decreased signalling) of the selected variants in IIS pathway components (GHRHR, GH1, IGF1, INS, IRS1), we calculated composite IIS scores to estimate IIS pathway activity. In addition, we analysed the relative impact on lifespan and body size of the separate variants in multivariate models. In women, lower IIS scores are significantly associated with lower body height and improved old age survival. Multivariate analyses showed that these results were most pronounced for the GH1 SNP, IGF1 CA repeat and IRS1 SNP. In females, for variant allele carriers of the GH1 SNP, body height was 2 cm lower (P = 0.007) and mortality 0.80-fold reduced (P = 0.019) when compared with wild-type allele carriers. Thus, in females, genetic variation causing reduced IIS activation is beneficial for old age survival. This effect was stronger for the GH1 SNP than for variation in the conserved IIS genes that were found to affect longevity in model organisms.

β-Glucosidases (3.2.1.21) are found in all domains of living organisms, where they play essential roles in the removal of nonreducing terminal glucosyl residues from saccharides and glycosides. β-Glucosidases function in glycolipid and exogenous glycoside metabolism in animals, defense, cell wall lignification, cell wall β-glucan turnover, phytohormone activation, and release of aromatic compounds in plants, and biomass conversion in microorganisms. These functions lead to many agricultural and industrial applications. β-Glucosidases have been classified into glycoside hydrolase (GH) families GH1, GH3, GH5, GH9, and GH30, based on their amino acid sequences, while other β-glucosidases remain to be classified. The GH1, GH5, and GH30 β-glucosidases fall in GH Clan A, which consists of proteins with (β/α)(8)-barrel structures. In contrast, the active site of GH3 enzymes comprises two domains, while GH9 enzymes have (α/α)(6) barrel structures. The mechanism by which GH1 enzymes recognize and hydrolyze substrates with different specificities remains an area of intense study.

Despite the key role of the linker histone H1 in chromatin structure and dynamics, its location and interactions with nucleosomal DNA have not been elucidated. In this work we have used a combination of electron cryomicroscopy, hydroxyl radical footprinting, and nanoscale modeling to analyze the structure of precisely positioned mono-, di-, and trinucleosomes containing physiologically assembled full-length histone H1 or truncated mutants of this protein. Single-base resolution *OH footprinting shows that the globular domain of histone H1 (GH1) interacts with the DNA minor groove located at the center of the nucleosome and contacts a 10-bp region of DNA localized symmetrically with respect to the nucleosomal dyad. In addition, GH1 interacts with and organizes about one helical turn of DNA in each linker region of the nucleosome. We also find that a seven amino acid residue region (121-127) in the COOH terminus of histone H1 was required for the formation of the stem structure of the linker DNA. A molecular model on the basis of these data and coarse-grain DNA mechanics provides novel insights on how the different domains of H1 interact with the nucleosome and predicts a specific H1-mediated stem structure within linker DNA.

Thyroid or glucocorticoid hormone increases the synthesis of growth hormone (GH) by clonal lines of rat pituitary tumor cells. To investigate whether these increases arise from increased accumulation of GH-specific RNA sequences in the cytoplasm and nuclei of these cells, we adapted two existing procedures so that a 32P-labeled hybrid plasmid containing a cDNA sequence could be used to quantitate relative concentrations of the corresponding mRNA. One method (RNA gel blot hybridization) used electrophoresis of RNA, transfer to nitrocellulose paper, and hybridization to 32P-labeled plasmid. The other (RNA dot hybridization) used covalent attachment of RNA to activated cellulose paper squares and hybridization to 32P-labeled plasmid. As probe, we used a hybrid plasmid (pBR322-GH1) which we show by restriction analysis to contain a DNA sequence coding for rat GH. The results were comparable from both techniques and showed that incubation of GH3 cells with a thyroid hormone (triiodothyronine), a glucocorticoid hormone (dexamethasone), or both hormones caused an increase of cytoplasmic pre-GH mRNA sequences of about 4-, 22-, and 13-fold, respectively. Results obtained with the RNA gel blot hybridization method showed that hormonal stimulation leads to the induction of a single 1.0-kilobase species of pre-GH mRNA in the cytoplasm and of 2.7- and 1.0- kilobase species of GH-specific RNA in the nucleus.

Hyperkalemic periodic paralysis (HYPP) is an autosomal dominant disorder characterized by episodes of muscle weakness due to depolarization of the muscle cell membrane associated with elevated serum potassium. Electrophysiological studies have implicated the adult muscle sodium channel. Here, portions of the adult muscle sodium channel alpha-subunit gene were cloned and mapped near the human growth hormone locus (GH1) on chromosome 17. In a large pedigree displaying HYPP with myotonia, these two loci showed tight linkage to the genetic defect with no recombinants detected. Thus, it is likely that the sodium channel alpha-subunit gene contains the HYPP mutation.

The nature of the complexes of histones H1 and H5 and their globular domains (GH1 and GH5) with DNA suggested two DNA-binding sites which are likely to be the basis of the preference of H1 and H5 for the nucleosome, compared with free DNA. More recently the X-ray and NMR structures of GH5 and GH1, respectively, have identified two basic clusters on opposite sides of the domains as candidates for these sites. Removal of the positive charge at either location by mutagenesis impairs or abolishes the ability of GH5 to assemble cooperatively in 'tramline' complexes containing two DNA duplexes, suggesting impairment or loss of its ability to bind two DNA duplexes. The mutant forms of GH5 also fail to protect the additional 20 bp of nucleosomal DNA that are characteristically protected by H1, H5 and wild-type recombinant GH5. They still bind to H1/H5-depleted chromatin, but evidently inappropriately. These results confirm the existence of, and identify the major components of, two DNA-binding sites on the globular domain of histone H5, and they strongly suggest that both binding sites are required to position the globular domain correctly on the nucleosome.

A type-specific monoclonal antibody, LP10, precipitated a glycoprotein with a molecular weight of approximately 59,000 from purified herpes simplex virus type 1. Although this glycoprotein was similar in size to glycoprotein D (gD), it was shown to be less abundant in both virions and infected cells, to migrate more rapidly in its precursor form, to incorporate glucosamine but not mannose, and to have a more stable precursor in tunicamycin-treated cells than the gD precursor (pgD). Immunoassays of cells infected with insertion recombinants and intertypic recombinants localized the gene coding for the target antigen of LP10 to the unique short (Us) region at map units 0.892 to 0.924 excluding gD. The target antigen of LP10 was then definitively mapped to the Us4 open reading frame by immunoprecipitation of a polypeptide synthesized by in vitro translation of a Us4-specific transcript prepared by using an SP6 cloning This newly identified glycoprotein product of the Us4 gene of herpes simplex virus type 1 is distinct from the previously identified gB1, gC1, gE1, and gH1.

GH insensitivity (GHI) presents in childhood as growth failure and in its severe form is associated with dysmorphic and metabolic abnormalities. GHI may be caused by genetic defects in the GH-IGF-I axis or by acquired states such as chronic illness. This article discusses the former category. The field of GHI due to mutations affecting GH action has evolved considerably since the original description of the extreme phenotype related to homozygous GH receptor (GHR) mutations over 40 yr ago. A continuum of genetic, phenotypic, and biochemical abnormalities can be defined associated with clinically relevant defects in linear growth. The role and mechanisms of the GH-IGF-I axis in normal human growth is discussed, followed by descriptions of mutations in GHR, STAT5B, PTPN11, IGF1, IGFALS, IGF1R, and GH1 defects causing bioinactive GH or anti-GH antibodies. These defects are associated with a range of genetic, clinical, and hormonal characteristics. Genetic abnormalities causing growth failure that is less severe than the extreme phenotype are emphasized, together with an analysis of height and serum IGF-I across the spectrum of different types of GHR defects. An overall view of genotype and phenotype relationships is presented, together with an updated approach to the assessment of the patient with GHI, focusing on investigation of the GH-IGF-I axis and relevant molecular studies contributing to this diagnosis.

Although most cases of short stature associated with growth hormone (GH) deficiency are sporadic and idiopathic, some 5-30% have an affected first degree relative consistent with a genetic aetiology for the condition. Several different types of mutational lesion in the pituitary-expressed growth hormone (GH1) gene have been described in affected individuals. This review focuses primarily on the GH1 mutational spectrum and its unusual features, discusses potential mechanisms of mutagenesis and pathogenesis, and examines the correlation between mutant genotype and clinical phenotype. The characterization of pathological lesions in several other pituitary-expressed genes that are epistatic to GH1 (POU1F1, PROP1 and GHRHR) has identified additional causes of GH deficiency, the molecular genetics of which are also explored.

The UL74 (glycoprotein O [gO])-UL75 (gH)-UL115 (gL) complex of human cytomegalovirus (CMV), known as the gCIII complex, is likely to play an important role in the life cycle of the virus. The gH and gL proteins have been associated with biological activities, such as the induction of virus-neutralizing antibody, cell-virus fusion, and cell-to-cell spread of the virus. The sequences of the two gH gene variants, readily recognizable by restriction endonuclease polymorphism, are well conserved among clinical isolates, but nothing is known about the sequence variability of the gL and gO genes. Sequencing of the full-length gL and gO genes was performed with 22 to 39 clinical isolates, as well as with laboratory strains AD169, Towne, and Toledo, to determine phylogenetically based variants of the genes. The sequence information provided the basis for identifying gL and gO variants by restriction endonuclease polymorphism. The predicted gL amino acid sequences varied less than 2% among the isolates, but the variability of gO among the isolates approached 45%. The variants of the genes coding for gCIII in laboratory strains Towne, AD169, and Toledo were different from those in most clinical isolates. When clinical isolates from different patient populations with various degrees of symptomatic CMV disease were surveyed, the gO1 variant occurred almost exclusively with the gH1 variant. The gL2 variant occurred with a significantly lower frequency in the gH1 variant group. There were no configurations of the gCIII complex that were specifically associated with symptomatic CMV disease or human immunodeficiency virus serologic status. The potential for the gCIII complex to exist in diverse genetic combinations in clinical isolates points to a new aspect that must be considered in studies of the significance of CMV strain variability.

The worldwide incidence of hepatocellular carcinoma (HCC) is approximately one million cases a year. This makes HCC one of the most frequent human malignancies, especially in Asia and Africa, although the incidence is increasing also in the western world. HCC is a complication of chronic liver disease, with cirrhosis as the most important risk factor. Viral co-pathogenesis makes cirrhosis due to hepatitis B (HBV) and hepatitis C virus (HCV) infection a very important factor in the development of HCC. As curative therapy is often ruled out due to the late detection of HCC, it would be attractive to find parameters which predict malignant transformation in HBV- and HCV-infected livers. This study has used comparative genomic hybridization (CGH) to analyse 26 HCCs (11 non-viral, nine HBV, six HCV) and 12 concurrent dysplasias (five non-viral, five HBV, two HCV). Frequent gain >> or =25% of all tumours) was detected, in decreasing order of frequency, on 8q (69%), 1q (46%), 17q (46%), 12q (42%), 20q (31%), 5p (27%), 6q (27%), and Xq (27%). Frequent loss >> or =25% of all tumours) was found, in decreasing order of frequency, on 8p (58%), 16q (54%), 4q (42%), 13q (39%), 1p (35%), 4p (35%), 16p (35%), 18q (35%), 14q (31%), 17p (31%), 9p (27%), and 9q (27%). Minimal overlapping regions could be determined at multiple locations (candidate genes in parentheses). Minimal regions of overlap for deletions were assigned to 4p14-15 (PCDH7), 8p21-22 (FEZ1), 9p12-13, 13q14-31 (RB1), 14q31 (TSHR), 16p12-13.1 (GSPT1), 16q21-23 (CDH1), 17p12-13 (TP53), and 18q21-22 (DPC4, DCC). Minimal overlapping amplified sites could be seen at 8q24 (MYC), 12q15-21 (MDM2), 17q22-25 (SSTR2, GH1), and 20q12-13.2 (MYBL2, PTPN1). A single high level amplification was seen on 5q21 in an HBV-related tumour. Aberrations appeared more frequent in HBV-related HCCs than in HCV-associated tumours (p=0.008). This was most prominent with respect to losses (p=0.004), specifically loss on 4p (p=0.007), 16q (p=0.04), 17p (p=0.04), and 18q (p=0.03). In addition, loss on 17p was significantly lower in non-viral cancers than in HBV-related HCC (p<0.001). Furthermore, loss on 13q was more prevalent in HCCs in non-cirrhotic livers (p=0.02), thus suggesting a different, potentially more aggressive, pathway in neoplastic progression. A tendency (p=0.07) was observed for loss on 9q in high-stage tumours; no specific changes were found in relation to tumour grade. A subset of the HCC-associated genetic changes was disclosed in the preneoplastic stage, i.e. liver cell dysplasia. This group of dysplasias showed frequent gain on 17q (25%) and frequent loss on 16q (33%), 4q (25%), and 17p (25%). The majority of the dysplasias with alterations revealed genetic changes that were also present in the primary tumour. In conclusion, firstly, this study has provided a detailed map of genomic changes occurring in HCC of viral and non-viral origin, and has suggested candidate genes. Loss on 17p, including the TP53 region, appeared significantly more prevalent in HBV-associated liver cancers, whereas loss on 13q, with possible involvement of RB1, was distinguished as a possible genetic biomarker. Secondly, CGH analysis of liver cell dysplasia, both viral and non-viral, has revealed HCC-specific early genetic changes, thereby confirming its preneoplastic nature. Finally, genes residing in these early altered regions, such as CDH1 or TP53, might be associated with hepatocellular carcinogenesis.

We previously reported that putative nuclear receptors for thyroid hormone can be demonstrated by incubation of hormone either with intact GH(1) cells, a rat pituitary tumor cell line, or with isolated GH(1) cell nuclei and rat liver nuclei in vitro. We characterized further the kinetics of triiodothyronine (T3) and thyroxine (T4) binding and the biochemical properties of the nuclear receptor after extraction to a soluble form with 0.4 M KCl. In vitro binding of [(125)I]T3 and [(125)I]T4 with GH(1) cell and rat liver nuclear extract was examined at 0 degrees C and 37 degrees C. Equilibrium was attained within 5 min at 37 degrees C and 2 h at 0 degrees C. The binding activity from GH(1) cells was stable for at least 1 h at 37 degrees C and 10 days at - 20 degrees C. Chromatography on a weak carboxylic acid column and inactivation by trypsin and Pronase, but not by DNase or RNase, suggested that the putative receptor was a nonhistone protein. The estimated equilibrium dissociation constants (K(d)) for hormone binding to the solubilized nuclear binding activity was 1.80 x 10(-10) M (T3) and 1.20 x 10(-9) M (T4) for GH(1) cells and 1.57 x 10(-10) M (T3) and 2.0 x 10(-9) M (T4) for rat liver. These K(d) values for T3 are virtually identical to those which we previously reported with isolated rat liver nuclei and GH(1) cell nuclei in vitro. The 10-fold greater affinity for T3 compared to T4 in the nuclear extract is also identical to that observed with intact GH(1) cells. In addition, the [(125)I]T3 and [(125)I]T4 high-affinity binding in the nuclear extract were inhibited by either nonradioactive T3 or T4, which suggests that the binding activity in nuclear extract was identical for T3 and T4. In contrast, the binding activity for T4 and T3 in GH(1) cell cytosol was markedly different from that observed with nuclear extract (K(d) values were 2.87 x 10(-10) M for T4 and 1.13 x 10(-9) M for T3). Our results indicate that nuclear receptors for T3 and T4 can be isolated in a soluble and stable form with no apparent change in hormonal affinity. This should allow elucidation of the mechanisms of thyroid hormone action at the molecular level.

Neutron diffraction data from a crystal of carbonmonoxymyoglobin were refined by PROLSQ, a modern restrained least-squares procedure in reciprocal space, in conjunction with a solvent analysis technique, to a final R-factor of 11.3%. The ligand CO occupies two sites and its binding conformations are distorted from the linear conformation. The N epsilon atom of the distal histidine residue is deprotonated (not deuterated), and a water molecule is bound to the N delta atom of the distal histidine. The side-chain of Lys56 (D6) exists in two alternative charge-binding sites. His24 (B5) and His119 (GH1) share a hydrogen atom. His12 (A10) and His36 (C1) are deprotonated. The deprotonated imidazole ring of His12 (A10) may act as a hydrogen-bond acceptor. The heme group is planar within 0.09 A root-mean-square (r.m.s.) deviation from planarity. The solvent environments for the two propionic acid groups are different. The side-chain of Arg45 (CD3) forms hydrogen bonds with the side-chain of Asp60 (E3) and one of the two propionic acid groups. An average N-2H . . . O angle in helical regions is 147 (+/- 11) degrees. Eleven main-chain amide hydrogen atoms from hydrophobic residues do not exchange with deuterium. The overall atomic occupancy factors for the main-chain and side-chain atoms are quite uniform, at 0.97 (+/- 0.07) and 0.93 (+/- 0.10), respectively, as shown by an occupancy analysis made at the end of the refinement procedure.

The proximal promoter region of the human pituitary expressed growth hormone (GH1) gene is highly polymorphic, containing at least 15 single nucleotide polymorphisms (SNPs). This variation is manifest in 40 different haplotypes, the high diversity being explicable in terms of gene conversion, recurrent mutation, and selection. Functional analysis showed that 12 haplotypes were associated with a significantly reduced level of reporter gene expression whereas 10 haplotypes were associated with a significantly increased level. The former tend to be more prevalent in the general population than the latter (p<0.01), possibly as a consequence of selection. Although individual SNPs contributed to promoter strength in a highly interactive and non-additive fashion, haplotype partitioning was successful in identifying six SNPs as major determinants of GH1 gene expression. The prediction and functional testing of hitherto unobserved super-maximal and sub-minimal promoter haplotypes was then used to test the efficacy of the haplotype partitioning approach. Electrophoretic mobility shift assays demonstrated that five SNP sites exhibit allele-specific protein binding. An association was noted between adult height and the mean in vitro expression value corresponding to an individual's GH1 promoter haplotype combination (p=0.028) although only 3.3% of the variance of adult height was found to be explicable by reference to this parameter. Three additional SNPs, identified within sites I and II of the upstream locus control region (LCR), were ascribed to three distinct LCR haplotypes. A series of LCR-GH1 proximal promoter constructs were used to demonstrate that 1) the LCR enhanced proximal promoter activity by up to 2.8-fold depending upon proximal promoter haplotype, and that 2) the activity of a given proximal promoter haplotype was also differentially enhanced by different LCR haplotypes. The genetic basis of inter-individual differences in GH1 gene expression thus appears to be extremely complex.

The globular domain of chicken histone H1 (GH1) has been studied by 1H homonuclear and 1H-15N heteronuclear 2D NMR spectroscopy. After the full assignment of the proton and 15N resonances, the tertiary structure of GH1 was determined by an iterative procedure using distance geometry and restrained simulated annealing. The secondary structure elements of GH1, three helices (S5-A16, S24-A34, N42-K56) followed by a beta-hairpin (L59-L73), are folded in a manner very similar to the corresponding parts of the globular domain of chicken histone H5 (GH5) [Clore et al. (1987) EMBO J. 6, 1833-1842; Ramakrishnan et al. (1993) Nature 362, 219-223]. However, subtle differences are detected between the two structures and between the electrostatic potentials surrounding the molecules. The most important differences are located in the loop between the second and third helices, a region that could be responsible for the different affinity for DNA. The most positively charged regions are not found in exactly the same position in GH1 and GH5. Nevertheless, their location seems to agree with the model where nucleosome binding takes place through contact points located at one DNA terminus and close to the dyad axis of the nucleosome [Schwabe & Travers (1993) Curr. Biol. 3, 628-630].

The construction, identification, and use of a recombinant DNA clone containing a growth hormone structural gene sequence is described. A cDNA copy of partially purified pregrowth hormone mRNA from cultured rat pituitary tumor (GC) cells was employed in the construction of a hybrid plasmid, designated pBR322-GH1. The cloned DNA sequence was positively identified by a hybridization-translation procedure which should be applicable to any cloned structural gene sequence. This procedure involved hybridization of cytoplasmic poly(A)-containing RNA from GC cells to the cloned DNA immobilized on nitrocellulose filters, followed by elution of the hybridized RNA and translation in a mRNA-depleted rabbit reticulocyte lysate system. Physical and immunological criteria were employed to show that the translation products were enriched for pregrowth hormone. Hybridization to excess plasmid DNA of [3H]uridine-labeled, size fractionated GC cell cytoplasmic RNA was used to show that all growth hormone-specific RNA sequences are the same size as functional pregrowth hormone mRNA.

In view of the likely role of H1-H1 interactions in the stabilization of chromatin higher order structure, we have asked whether interactions can occur between the globular domains of the histone molecules. We have studied the properties of the isolated globular domains of H1 and the variant H5 (GH1 and GH5) and we have shown (by sedimentation analysis, electron microscopy, chemical cross-linking and nucleoprotein gel electrophoresis) that although GH1 shows no, and GH5 little if any, tendency to self-associate in dilute solution, they bind highly cooperatively to DNA. The resulting complexes appear to contain essentially continuous arrays of globular domains bridging 'tramlines' of DNA, similar to those formed with intact H1, presumably reflecting the ability of the globular domain to bind more than one DNA segment, as it is likely to do in the nucleosome. Additional (thicker) complexes are also formed with GH5, probably resulting from association of the primary complexes, possibly with binding of additional GH5. The highly cooperative nature of the binding, in close apposition, of GH1 and GH5 to DNA is fully compatible with the involvement of interactions between the globular domains of H1 and its variants in chromatin folding.

Using cultured GH1 cells, we reported that stimulation (3- to 5-fold) of growth hormone synthesis and mRNA levels by thyroid hormone is mediated by a chromatin-associated receptor. Thyroid hormone also elicits a rapid reduction of homologous receptor in GH1 cells primarily by decreasing the synthetic rate of receptor ( Raaka , B. M., and Samuels , H. H. (1981) J. Biol. Chem. 256, 6883-6889). Without 3,5,3'-triiodo-L-thyronine (L-T3), glucocorticoid agonists induced a limited and delayed effect while L-T3 + glucocorticoid synergistically stimulated the response an additional 2- to 4-fold compared to L-T3. In this study, we utilized GC cells, a related cell line, to compare the abundance of L-T3-receptor complexes to the rate of growth hormone mRNA synthesis and gene transcription. Gene transcription was assessed by in vitro labeling of nuclei with [alpha-32P]UTP which were derived from cells incubated with hormone(s), while mRNA synthesis was determined in intact cells by [3H]uridine labeling. Labeled growth hormone mRNA and gene transcripts were quantitated by filter hybridization to plasmid containing growth hormone cDNA. L-T3 rapidly decreased receptor levels in GC cells with kinetics similar to that in GH1 cells. Both the L-T3 and the synergistic L-T3 + glucocorticoid stimulation of growth hormone mRNA synthesis changed in parallel with the level of L-T3-receptor complexes. Glucocorticoid hormones alone elicited a variable response which resulted in minimal stimulation or inhibition of growth hormone mRNA synthesis or gene transcription rates. No apparent lag was identified between the kinetics of L-T3 binding to receptor and stimulation of growth hormone gene transcription. L-T3 stimulated growth hormone gene transcription rates maximally in 1 h which then progressively decreased in parallel with L-T3-receptor levels. Using [3H]uridine pulse-chase, growth hormone mRNA was found to have a half-life of approximately 50 h in agreement with the decay curve of growth hormone production of deinduced cells. Our studies suggest that regulation of the growth hormone response is predominantly determined by positive control of growth hormone gene transcription which is proportional to the concentration of thyroid hormone-receptor complexes.

BACKGROUND

Since the times of domestication, cattle have been continually shaped by the influence of humans. Relatively recent history, including breed formation and the still enduring enormous improvement of economically important traits, is expected to have left distinctive footprints of selection within the genome. The purpose of this study was to map genome-wide selection signatures in ten cattle breeds and thus improve the understanding of the genome response to strong artificial selection and support the identification of the underlying genetic variants of favoured phenotypes. We analysed 47,651 single nucleotide polymorphisms (SNP) using Cross Population Extended Haplotype Homozygosity (XP-EHH).

RESULTS

We set the significance thresholds using the maximum XP-EHH values of two essentially artificially unselected breeds and found up to 229 selection signatures per breed. Through a confirmation process we verified selection for three distinct phenotypes typical for one breed (polledness in Galloway, double muscling in Blanc-Bleu Belge and red coat colour in Red Holstein cattle). Moreover, we detected six genes strongly associated with known QTL for beef or dairy traits (TG, ABCG2, DGAT1, GH1, GHR and the Casein Cluster) within selection signatures of at least one breed. A literature search for genes lying in outstanding signatures revealed further promising candidate genes. However, in concordance with previous genome-wide studies, we also detected a substantial number of signatures without any yet known gene content.

CONCLUSIONS

These results show the power of XP-EHH analyses in cattle to discover promising candidate genes and raise the hope of identifying phenotypically important variants in the near future. The finding of plausible functional candidates in some short signatures supports this hope. For instance, MAP2K6 is the only annotated gene of two signatures detected in Galloway and Gelbvieh cattle and is already known to be associated with carcass weight, back fat thickness and marbling score in Korean beef cattle. Based on the confirmation process and literature search we deduce that XP-EHH is able to uncover numerous artificial selection targets in subpopulations of domesticated animals.

The vectorial transport of vesicular stomatitis virus (VSV) G protein between the ER and the cis and medial Golgi compartments has been reconstituted using semi-intact (perforated) cells. The transport of VSV-G protein between successive compartments is measured by the sequential processing of the two N-linked oligosaccharide chains present on VSV-G protein to the endoglycosidase (endo) H-resistant structures which have unique electrophoretic mobilities during sodium dodecyl sulfate-gel electrophoresis. The appearance of a form of VSV-G which contains only one endo H-resistant oligosaccharide chain (GH1) is kinetically and biochemically indistinguishable from the appearance of the Man5, endo D-sensitive form (GD), the latter being a processing reaction diagnostic of transport from the ER to the cis Golgi compartment. These results provide evidence that the cis Golgi compartment may contain in addition to alpha-1,2-mannosidase I, both N-acetylglueosamine transferase I and alpha-1,2-mannosidase II. VSV-G protein is subsequently processed to the form which contains two endo H-resistant oligosaccharides (GH2) after a second wave of vesicular transport. Processing of GH1 to GH2 in vitro occurs only after a lag period following the appearance of GH1; processing is sensitive to N-ethylmaleimide, guanosine-5'-O-(3-thiotriphosphate), and a synthetic peptide homologous to the rab1 protein effector domain, and processing is inhibited in the absence of free Ca2+ (in the presence of EGTA), reagents which potently inhibit ER to cis Golgi transport. These results suggest that VSV-G protein proceeds through at least two rounds of vesicular transport from the ER to the medial Golgi compartment for processing to the GH2 form, providing a model system to study the regulation of the vectorial membrane fission and fusion events involved in vesicular trafficking and organelle dynamics in the early stages of the secretory pathway.

BACKGROUND

Glycosyl hydrolase family 1 (GH1) beta-glucosidases have been implicated in physiologically important processes in plants, such as response to biotic and abiotic stresses, defense against herbivores, activation of phytohormones, lignification, and cell wall remodeling. Plant GH1 beta-glucosidases are encoded by a multigene family, so we predicted the structures of the genes and the properties of their protein products, and characterized their phylogenetic relationship to other plant GH1 members, their expression and the activity of one of them, to begin to decipher their roles in rice.

RESULTS

Forty GH1 genes could be identified in rice databases, including 2 possible endophyte genes, 2 likely pseudogenes, 2 gene fragments, and 34 apparently competent rice glycosidase genes. Phylogenetic analysis revealed that GH1 members with closely related sequences have similar gene structures and are often clustered together on the same chromosome. Most of the genes appear to have been derived from duplications that occurred after the divergence of rice and Arabidopsis thaliana lineages from their common ancestor, and the two plants share only 8 common gene lineages. At least 31 GH1 genes are expressed in a range of organs and stages of rice, based on the cDNA and EST sequences in public databases. The cDNA of the Os4bglu12 gene, which encodes a protein identical at 40 of 44 amino acid residues with the N-terminal sequence of a cell wall-bound enzyme previously purified from germinating rice, was isolated by RT-PCR from rice seedlings. A thioredoxin-Os4bglu12 fusion protein expressed in Escherichia coli efficiently hydrolyzed beta-(1,4)-linked oligosaccharides of 3-6 glucose residues and laminaribiose.

CONCLUSIONS

Careful analysis of the database sequences produced more reliable rice GH1 gene structure and protein product predictions. Since most of these genes diverged after the divergence of the ancestors of rice and Arabidopsis thaliana, only a few of their functions could be implied from those of GH1 enzymes from Arabidopsis and other dicots. This implies that analysis of GH1 enzymes in monocots is necessary to understand their function in the major grain crops. To begin this analysis, Os4bglu12 beta-glucosidase was characterized and found to have high exoglucanase activity, consistent with a role in cell wall metabolism.

We have previously demonstrated that thyroid hormone controls growth hormone synthesis in GH1 cells and that the induction of the growth hormone response by glucocorticoid appears to be highly dependent on thyroid hormone action. Thyroid hormone induces growth hormone synthesis approximately 5- to 20-fold and cortisol increases this response 2- to 6-fold further. Long-term kinetics of the growth hormone response show that, without added thyroid hormone, cortisol can induce a small-growth hormone response after 48 hr of incubation. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of proteins synthesized in intact cells demonstrates that the cortisol enhancement of growth hormone synthesis in cells incubated with thyroid hormone is a relatively selective process. Quantitation of growth hormone mRNA levels by cell-free protein synthesis demonstrates that the regulation of growth hormone synthesis by thyroid and glucocorticoid hormones is explained by a synergistic pretranslational control mechanism, presumably at the level of the growth hormone gene.

The bovine rumen represents a highly specialized bioreactor where plant cell wall polysaccharides (PCWPs) are efficiently deconstructed via numerous enzymes produced by resident microorganisms. Although a large number of fibrolytic genes from rumen microorganisms have been identified, it remains unclear how they are expressed in a coordinated manner to efficiently degrade PCWPs. In this study, we performed a metatranscriptomic analysis of the rumen microbiomes of adult Holstein cows fed a fiber diet and obtained a total of 1,107,083 high-quality non-rRNA reads with an average length of 483 nucleotides. Transcripts encoding glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs) accounted for 1% and 0.1% of the total non-rRNAs, respectively. The majority (98%) of the putative cellulases belonged to four GH families (i.e., GH5, GH9, GH45, and GH48) and were primarily synthesized by Ruminococcus and Fibrobacter. Notably, transcripts for GH48 cellobiohydrolases were relatively abundant compared to the abundance of transcripts for other cellulases. Two-thirds of the putative hemicellulases were of the GH1GH1GH1, GH2, GH3, and GH43 proteins and were from a diverse group of microorganisms. Transcripts for CBM10 and dockerin, key components of the cellulosome, were also relatively abundant. Our results provide metatranscriptomic evidence in support of the notion that members of the genera Ruminococcus, Fibrobacter, and Prevotella are predominant PCWP degraders and point to the significant contribution of GH48 cellobiohydrolases and cellulosome-like structures to efficient PCWP degradation in the cow rumen.