Amino Acid Limitation Research Articles

Glucose limitation induces GCN4 translation by activation of Gcn2 protein kinase.

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) is a well-characterized mechanism regulating protein synthesis in response to environmental stresses. In the yeast Saccharomyces cerevisiae, starvation for amino acids induces phosphorylation of eIF-2alpha by Gcn2 protein kinase, leading to elevated translation of GCN4, a transcriptional activator of more than 50 genes. Uncharged tRNA that accumulates during amino acid limitation is proposed to activate Gcn2p by associating with Gcn2p sequences homologous to histidyl-tRNA synthetase (HisRS) enzymes. Given that eIF-2alpha phosphorylation in mammals is induced in response to both carbohydrate and amino acid limitations, we addressed whether activation of Gcn2p in yeast is also controlled by different nutrient deprivations. We found that starvation for glucose induces Gcn2p phosphorylation of eIF-2alpha and stimulates GCN4 translation. Induction of eIF-2alpha phosphorylation by Gcn2p during glucose limitation requires the function of the HisRS-related domain but is largely independent of the ribosome binding sequences of Gcn2p. Furthermore, Gcn20p, a factor required for Gcn2 protein kinase stimulation of GCN4 expression in response to amino acid starvation, is not essential for GCN4 translational control in response to limitation for carbohydrates. These results indicate there are differences between the mechanisms regulating Gcn2p activity in response to amino acid and carbohydrate deficiency. Gcn2p induction of GCN4 translation during carbohydrate limitation enhances storage of amino acids in the vacuoles and facilitates entry into exponential growth during a shift from low-glucose to high-glucose medium. Gcn2p function also contributes to maintenance of glycogen levels during prolonged glucose starvation, suggesting a linkage between amino acid control and glycogen metabolism.

The stringent response in Myxococcus xanthus is regulated by SocE and the CsgA C-signaling protein

Myxococcus xanthus fruiting body development is induced by amino acid limitation. The decision to grow or develop is established by the RelA-dependent stringent response and A-signaling. We identified two new members of this regulatory hierarchy, socE and the C-signaling gene csgA. SocE depletion arrests growth and induces sporulation under conditions that normally favor growth as well as curtailing DNA and stable RNA synthesis, inhibiting cell elongation, and inducing accumulations of the stringent nucleotides ppGpp and pppGpp [(p)ppGpp]. This system separates C-signaling, which does not occur under these conditions, from CsgA enzyme activity. Amino acid substitutions in the CsgA coenzyme binding pocket or catalytic site eliminate growth arrest. relA mutation also eliminates growth arrest. Eleven pseudorevertants selected for growth following SocE depletion contained mutations in csgA or relA. These results suggest that CsgA induces the stringent response and while SocE inhibits it. Unlike the csgA mutant, wild-type and socE csgA cells maintained high levels of (p)ppGpp throughout development. We suggest that CsgA maintains growth arrest throughout development to divert carbon from A-signaling and other sources into developmental macromolecular synthesis.

A mammalian homologue of GCN2 protein kinase important for translational control by phosphorylation of eukaryotic initiation factor-2alpha.

A family of protein kinases regulates translation in response to different cellular stresses by phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2alpha). In yeast, an eIF-2alpha kinase, GCN2, functions in translational control in response to amino acid starvation. It is thought that uncharged tRNA that accumulates during amino acid limitation binds to sequences in GCN2 homologous to histidyl-tRNA synthetase (HisRS) enzymes, leading to enhanced kinase catalytic activity. Given that starvation for amino acids also stimulates phosphorylation of eIF-2alpha in mammalian cells, we searched for and identified a GCN2 homologue in mice. We cloned three different cDNAs encoding mouse GCN2 isoforms, derived from a single gene, that vary in their amino-terminal sequences. Like their yeast counterpart, the mouse GCN2 isoforms contain HisRS-related sequences juxtaposed to the kinase catalytic domain. While GCN2 mRNA was found in all mouse tissues examined, the isoforms appear to be differentially expressed. Mouse GCN2 expressed in yeast was found to inhibit growth by hyperphosphorylation of eIF-2alpha, requiring both the kinase catalytic domain and the HisRS-related sequences. Additionally, lysates prepared from yeast expressing mGCN2 were found to phosphorylate recombinant eIF-2alpha substrate. Mouse GCN2 activity in both the in vivo and in vitro assays required the presence of serine-51, the known regulatory phosphorylation site in eIF-2alpha. Together, our studies identify a new mammalian eIF-2alpha kinase, GCN2, that can mediate translational control.

Growth hormone and amino acid supply interact synergistically to control insulin-like growth factor-I production and gene expression in cultured ovine hepatocytes.

Many of the anabolic effects of growth hormone (GH) are indirect, occurring through GH-stimulated production of insulin-like growth factor-I (IGF-I) by the liver. As well as being regulated by GH, plasma IGF-I concentrations have been demonstrated to depend upon the level of dietary protein intake, with low protein diets being associated with reduced circulatory IGF-I levels. This inhibitory effect cannot be reversed by GH injection, suggesting that liver sensitivity to GH becomes impaired.To investigate the mechanisms through which protein supply affects GH sensitivity, primary cultures of ovine hepatocytes were grown in defined media, containing various proportions (0.2, 1.0 and 5.0) of jugular amino acid concentrations in fed sheep. Production of IGF-I by these cells was measured after 24 and 48 h in culture by radioimmunoassay. In the first 24-h period basal IGF-I production was the same in all defined media, and GH caused an approximately 2-fold increase in IGF-I release in cells grown in 1.0xor 5.0xamino acid media (P<0.01). Although GH appeared to increase IGF-I release in this period for cells grown in 0.2xamino acid media, this effect was not statistically significant. In the period from 24-48 h in defined media, both basal and GH-stimulated IGF-I production was dependent on amino acid availability (P<0.05 and P<0.001 respectively). Factorial analysis of variance demonstrated a strong positive interaction (P<0.001) between the effects of amino acid availability and GH, such that GH increased IGF-I production by more than 2-fold in cells grown in 5.0xamino acid media (P<0.01) but had no effect on production by cells grown in 1.0xor 0.2xamino acid media. Measurement of steady state concentrations of exon 1-derived IGF-I mRNAs using an RNase protection assay demonstrated that the observed effects on IGF-I peptide secretion were strongly associated with parallel effects at the RNA level. Incorporation of (35)S-methionine into cellular proteins over a 4-h period starting 20 h after transfer to defined culture media was not significantly reduced in 1.0xcompared with 5.0x amino acid media, although rates under both of these conditions were significantly higher than those seen in 0.2xamino acid media (P<0.01). The lack of correspondence between the dose-dependent effects of amino acid supply on cellular protein synthesis and those on basal and GH-stimulated IGF-I production, suggests that amino acid supply modulates IGF-I production through selective mechanisms. Steady state levels of the CCAAT/enhancer-binding protein beta (C/EBPbeta) isoforms, liver-enriched activating protein (LAP) and liver-enriched inhibitory protein (LIP) were determined by Western blotting. When levels of LAP were expressed relative to LIP levels in the same extracts, a significant decrease in the LAP:LIP ratio was observed in response to amino acid limitation (P<0.05). These data strengthen earlier arguments that synergistic interaction between the effects of amino acids and GH on hepatic IGF-I gene expression underlie nutrition-dependent changes in circulating IGF-I titres. The association between these effects and altered levels of C/EBPbeta isoforms suggests that CCAAT/enhancer mediated control of IGF-I gene expression may be involved in this phenomenon.

Functional activity of hepatocyte nuclear factor-1 is specifically decreased in amino acid-limited hepatoma cells

Limitation of cultured rat hepatoma cells for an essential amino acid results in a specific decrease in expression of several genes that are preferentially expressed in the liver, including the serum albumin and transthyretin genes. In the work presented here, we examined whether the coordinate repression of these genes is caused by decreased activity of one or more of the liver-enriched transcription factors, hepatocyte nuclear factor-1 (HNF-1), HNF-3, HNF-4 or C/EBP. To address this question, HepG2 human hepatoma cells were transiently transfected with luciferase reporter constructs containing multiple copies of individual transcription factor binding sites. Limitation for an essential amino acid resulted in specific repression of a construct in which luciferase expression was directed by HNF-1. A single HNF-1 binding site located adjacent to the TATA box plays a major role in transcription directed by the serum albumin promoter in transient transfection assays. Amino acid limitation of cells transfected with an albumin promoter/luciferase reporter construct resulted in specific repression of promoter activity. In addition, bacterial methylation or site-directed mutagenesis of the HNF-1 binding site in the albumin proximal promoter region eliminated the regulation of an albumin promoter-luciferase reporter construct under conditions of amino acid limitation. These results demonstrated that the HNF-1 binding site played a major role in regulation of the albumin promoter by amino acid availability. Deletion analysis of the albumin promoter confirmed regulation through the HNF-1 binding site and also identified a second amino acid regulatory element in the upstream region of the albumin promoter, which has been shown previously to contain a functional binding site for HNF-3. The repression of albumin promoter and HNF-1 reporter constructs in amino acid-limited cells occurred without a change in the DNA binding activity of HNF-1. Moreover, HNF-3 DNA binding activity was also not decreased in amino acid-limited cells. These results suggest that the regulation of transcription by amino acids occurs at the level of transcriptional activation by HNF-1 and HNF-3, rather than by alteration of the DNA binding activity of either factor.

Amino acid limitation regulates gene expression.

In mammals, the plasma concentration of amino acids is affected by nutritional or pathological conditions. For example, an alteration in the amino acid profile has been reported when there is a deficiency of any one or more of the essential amino acids, a dietary imbalance of amino acids, or an insufficient intake of protein. We examined the role of amino acid limitation in regulating mammalian gene expression. Depletion of arginine, cystine and all essential amino acids leads to induction of insulin-like growth factor-binding protein-1 (IGFBP-1) mRNA and protein expression in a dose-dependent manner. Moreover, exposure of HepG2 cells to amino acids at a concentration reproducing the amino acid concentration found in portal blood of rats fed on a low-protein diet leads to a significantly higher (P < 0.0002) expression of IGFBP-1. Using CCAAT/enhancer-binding protein homologous protein (CHOP) induction by leucine deprivation as a model, we have characterized the molecular mechanisms involved in the regulation of gene expression by amino acids. We have shown that leucine limitation leads to induction of CHOP mRNA and protein. Elevated mRNA levels result from both an increase in the rate of CHOP transcription and an increase in mRNA stability. We have characterized two elements of the CHOP gene that are essential to the transcriptional activation produced by an amino acid limitation. These findings demonstrate that an amino acid limitation, as occurs during dietary protein deficiency, can induce gene expression. Thus, amino acids by themselves can play, in concert with hormones, an important role in the control of gene expression.

Amino acid regulation of gene expression.

In mammals, the plasma concentration of amino acids is affected by nutritional or pathological conditions. For example, an amino acid profile alteration has been reported as a result of a deficiency of any one of the essential amino acids, a dietary imbalance of amino acids or an insufficient intake of protein. Amino acid availability regulates the expression of several genes involved in the regulation of growth, cellular function or amino acid metabolism. A limitation of several amino acids strongly increases the expression of insulin-like growth factor binding protein CHOP and asparagine synthetase genes. Elevated messenger RNA levels result from both an increase in the rate of transcription and an increase in messenger RNA stability. DNA amino acid response elements have been characterized in the promoter of CHOP and asparagine synthetase genes. The underlying mechanisms of gene regulation by amino acid limitation are not yet completely understood. The results discussed in this review demonstrate that amino acids by themselves can play, in concert with hormones, an important role in the control of gene expression.

Characterization of the major superoxide dismutase of Staphylococcus aureus and its role in starvation survival, stress resistance, and pathogenicity.

A Staphylococcus aureus mutant (SPW1) which is unable to survive long-term starvation was shown to have a transposon insertion within a gene homologous to the sodA family of manganese-dependent superoxide dismutases (SOD). Whole-cell lysates of the parental 8325-4 strain demonstrated three zones of SOD activity by nondenaturing gel electrophoresis. The activities of two of these zones were dependent on manganese for activity and were absent in SPW1. The levels of SOD activity and sodA expression were growth-phase dependent, occurring most during postexponential phase. This response was also dependent on the level of aeration of the culture, with highest activity and expression occurring only under high aeration. Expression of sodA and, consequently, SOD activity could be induced by methyl viologen but only during the transition from exponential- to postexponential-phase growth. SPW1 was less able to survive amino acid limitation and acid stress but showed no alteration in pathogenicity in a mouse abscess model of infection compared to the parental strain 8325-4.

SSB, encoding a ribosome-associated chaperone, is coordinately regulated with ribosomal protein genes.

Genes encoding ribosomal proteins and other components of the translational apparatus are coregulated to efficiently adjust the protein synthetic capacity of the cell. Ssb, a Saccharomyces cerevisiae Hsp70 cytosolic molecular chaperone, is associated with the ribosome-nascent chain complex. To determine whether this chaperone is coregulated with ribosomal proteins, we studied the mRNA regulation of SSB under several environmental conditions. Ssb and the ribosomal protein rpL5 mRNAs were up-regulated upon carbon upshift and down-regulated upon amino acid limitation, unlike the mRNA of another cytosolic Hsp70, Ssa. Ribosomal protein and Ssb mRNAs, like many mRNAs, are down-regulated upon a rapid temperature upshift. The mRNA reduction of several ribosomal protein genes and Ssb was delayed by the presence of an allele, EXA3-1, of the gene encoding the heat shock factor (HSF). However, upon a heat shock the EXA3-1 mutation did not significantly alter the reduction in the mRNA levels of two genes encoding proteins unrelated to the translational apparatus. Analysis of gene fusions indicated that the transcribed region, but not the promoter of SSB, is sufficient for this HSF-dependent regulation. Our studies suggest that Ssb is regulated like a core component of the ribosome and that HSF is required for proper regulation of SSB and ribosomal mRNA after a temperature upshift.

Amino acid limitation regulates CHOP expression through a specific pathway independent of the unfolded protein response

The gene encoding CHOP (C/EBP-homologous protein) is transcriptionally activated by many stimuli and by amino acid deprivation. CHOP induction was considered to be due to an accumulation of unfolded protein into the ER (unfolded protein response (UPR)). We investigate the role of the UPR in the induction of CHOP by amino acid deprivation and show that this induction is not correlated with BiP expression (an UPR marker). Moreover, amino acid deprivation and UPR inducers regulate the CHOP promoter activity using distinct cis elements. We conclude that amino acid deprivation does not activate the UPR and regulates CHOP expression through a pathway that is independent of the UPR.

The effects of amino acid supply on total and individual cellular protein synthesis in ovine hepatocytes

Protein nutrition, through altered amino-acid supply, regulates the quality and quantity of animal products, and deficiencies in supply of / response to amino-acids may be a limiting factor in ruminant efficiency. The effects of amino-acid supply may be indirect, via modulation of anabolic hormonal signalling. In particular, amino-acid limitation reduces insulin-like growth factor-1 (IGF-1) - and increases IGF-1 binding protein 1 (IGFBP-1) - production by the liver, and hence modulates the sensitivity of tissues to pituitary GH as an anabolic signal (Gluckman et al, 1987). The mechanisms through which amino-acids exert these selective effects on liver protein production are not understood.The present study sought to determine whether the effects of amino-acids on total cellular protein synthesis are sufficient to account for observed reductions in IGF-1 production in cultured ovine hepatocytes. In addition the use of two dimensional polyacrylamide gel electrophoresis (2D-PAGE) to visualise changes in translation of individual proteins caused by amino-acids in cultured ovine hepatocytes is described.

The yvyD gene of Bacillus subtilis is under dual control of sigmaB and sigmaH.

During a search by computer-aided inspection of two-dimensional (2D) protein gels for sigmaB-dependent general stress proteins exhibiting atypical induction profiles, a protein initially called Hst23 was identified as a product of the yvyD gene of Bacillus subtilis. In addition to the typical sigmaB-dependent, stress- and starvation-inducible pattern, yvyD is also induced in response to amino acid depletion. By primer extension of RNA isolated from the wild-type strain and appropriate mutants carrying mutations in the sigB and/or spo0H gene, two promoters were mapped upstream of the yvyD gene. The sigmaB-dependent promoter drives expression of yvyD under stress conditions and after glucose starvation, whereas a sigmaH-dependent promoter is responsible for yvyD transcription following amino acid limitation. Analysis of Northern blots revealed that yvyD is transcribed monocistronically and confirmed the conclusions drawn from the primer extension experiments. The analysis of the protein synthesis pattern in amino acid-starved wild-type and relA mutant cells showed that the YvyD protein is not synthesized in the relA mutant background. It was concluded that the stringent response plays a role in the activation of sigmaH. The yvyD gene product is homologous to a protein which might modify the activity of sigma54 in gram-negative bacteria. The expression of a sigmaL-dependent (sigmaL is the equivalent of sigma54 in B. subtilis) levD-lacZ fusion is upregulated twofold in a yvyD mutant. This indicates that the yvyD gene product, being a member of both the sigmaB and sigmaH regulons, might negatively regulate the activity of the sigmaL regulon. We conclude that (i) systematic, computer-aided analysis of 2D protein gels is appropriate for the identification of genes regulated by multiple transcription factors and that (ii) YvyD might form a junction between the sigmaB and sigmaH regulons on one side and the sigmaL regulon on the other.

Physiological concentration of amino acids regulates insulin-like-growth-factor-binding protein 1 expression.

Protein undernutrition is characterized by growth failure in young growing animals. Current evidence suggests that biosynthesis of insulin-like growth factor (IGF)-I and IGF-binding protein 1 (IGFBP-1) are key control points for nutritional regulation of growth. Here we examined the role of amino acid limitation in regulating the IGFBP-1 expression in the hepatic cell line. Our data show that leucine limitation strongly induces IGFBP-1 without affecting IGF-I and IGF-II expression in human HepG2 cells and in isolated rat hepatocytes. Depletion of arginine, cystine and all essential amino acids leads to induction of IGFBP-1 mRNA and protein expression in a dose-dependent manner. IGFBP-1 expression is significantly induced by leucine concentration in the range of that observed in the blood of rats fed a low-protein diet or in humans affected by kwashiorkor. Moreover, treatment of HepG2 cells with amino acids at a concentration reproducing the amino acid concentration found in portal blood of rats fed a low-protein diet leads to a significantly higher expression of IGFBP-1. These data represent the first demonstration that an amino acid limitation, as occurs during dietary protein deficiency, induces IGFBP-1 expression in hepatic cells. Therefore, amino acids by themselves can play, in concert with hormones, an important role in the control of gene expression.

Characterization of the starvation-survival response of Staphylococcus aureus.

The starvation-survival response of Staphylococcus aureus as a result of glucose, amino acid, phosphate, or multiple-nutrient limitation was investigated. Glucose and multiple-nutrient limitation resulted in the loss of viability of about 99 to 99.9% of the population within 2 days. The remaining surviving cells developed increased survival potential, remaining viable for months. Amino acid or phosphate limitation did not lead to the development of a stable starvation-survival state, and cells became nonculturable within 7 days. For multiple-nutrient limitation, the development of the starvation-survival state was cell density dependent. Starvation survival was associated with a decrease in cell size and increase in resistance to acid shock and oxidative stress. There was no evidence for the formation of a viable but nonculturable state during starvation as demonstrated by flow cytometry. Long-term survival of cells was dependent on cell wall and protein biosynthesis. Analysis of [35S]methionine incorporation and labelled proteins demonstrated that differential protein synthesis occurred deep into starvation.

The guanosine nucleotide (p)ppGpp initiates development and A-factor production in myxococcus xanthus.

Guanosine 3'-di-5'-(tri)di-phosphate nucleotides [(p)ppGpp], synthesized in response to amino acid limitation, induce early gene expression leading to multicellular fruiting body formation in Myxococcus xanthus. A mutant (DK527) that fails to accumulate (p)ppGpp in response to starvation was found to be blocked in development prior to aggregation. By use of a series of developmentally regulated Tn5lac transcriptional fusion reporters, the time of developmental arrest in DK527 was narrowed to within the few hours of development, the period of starvation recognition. The mutant is also defective in the production of A-factor, an early extracellular cell-density signal. The relA gene from Escherichia coli, which encodes a ribosome-dependent (p)ppGpp synthetase, rescues this mutant. We also demonstrate that inactivation of the M. xanthus relA homolog blocks development and the accumulation of (p)ppGpp. Moreover, the wild-type allele of Myxococcus relA rescues DK527. These observations support a model in which accumulation of (p)ppGpp, in response to starvation, initiates the program of fruiting body development, including the production of A-factor.

Order of amino acid limitation in poultry by-product meal

1. Two chick assays (from 8 to 17 d posthatching) were conducted to determine the order of amino acid (AA) limitation in poultry by-product meal (PBPM) using AA deletion and addition assays. 2. In the deletion assay, the basal diet contained 155 g/kg crude protein (provided solely by PBPM) and was fortified with cystine, tryptophan, threonine, lysine, valine, isoleucine, histidine, methionine and phenylalanine to meet the Illinois ideal AA Pattern. Each AA was then deleted individually from the basal diet and its effect on growth assessed. 3. In the addition assay, various individual or combined additions of nine AAs were made to an unfortified 155 g/kg crude protein diet containing PBPM as the sole source of dietary crude protein. 4. The results from the deletion assay were in general agreement with those from the addition assay. Based on the combined results of the deletion and addition assays, the order of AA limitation in PBPM was 1) cystine, 2) tryptophan, 3) threonine and lysine, 5) valine, and 6) isoleucine and histidine with the degree of deficiency of valine, isoleucine and histidine being similar. The results also suggested that methionine may be slightly limiting in PBPM protein.

Stationary phase, amino acid limitation and recovery from stationary phase modulate the stability and translation of chloramphenicol acetyltransferase mRNA and total mRNA in Escherichia coli

The functional stability of the chloramphenicol acetyltransferase (cat) mRNA, as well as the functional stability of the total mRNA pool, change during the course of Escherichia coli culture growth. mRNA half-lives are long during lag phase, decrease during the exponential phase and increase again during the stationary phase of the bacterial growth cycle. The half-lives of cat mRNA and total mRNA also increase three- to fourfold during amino acid starvation when compared to exponential culture growth. Even though the stability of the cat message changes about fourfold during culture growth, the amount of cat mRNA per cell mass does not vary significantly between the culture growth phases, indicating that there are compensating changes in cat gene transcription. Translation of cat mRNA also changes during culture growth. In exponential phase, the rate of cat translation is about 14-fold higher than when the culture is in stationary phase. This is in contrast to the fourfold increase in stability of cat mRNA in the stationary-phase culture compared to the exponentially growing culture and indicates that active translation is not correlated with increased mRNA stability. When a stationary-phase culture was diluted into fresh medium, there was a five- to sevenfold increase in CAT synthesis and a threefold increase in total protein synthesis in the presence or absence of rifampicin. These results suggest that while mRNA becomes generally more stable and less translated in the stationary-phase culture, the mRNA is available for immediate translation when nutrients are provided to the culture even when transcription is inhibited.

Effect of dietary methionine supplementation on hoof horn growth in primiparous cows

Horn growth rate does not appear to be related to the amino acid profile of the major protein source for dairy cattle (Offer, Logue &amp; Roberts, 1997), but it is possible that sulphur amino acids are limiting in early lactation, when the homeorhetic drive to milk production is most extreme. Supplementation of a high production ration for dairy cows with protected methionine should increase milk production, and allow any sulphur amino acid limitation on horn growth to be alleviated (Mengal, Galbraith, Souri &amp; Scaife, 1997).A total of 60 in-calf Holstein heifers were divided into two groups in a randomised block design. The study commenced approximately three weeks pre-calving until 26 weeks post calving, and animals were housed in one of three systems for the duration. Two diets were formulated based on a grass silage:maize silage mixture (50:50 DM basis), with rolled wheat, soya bean meal, sugar beet feed and rapeseed meal and were offered from approximately five days post calving for the remainder of the 26 week period.

Ribosome-binding Domain of Eukaryotic Initiation Factor-2 Kinase GCN2 Facilitates Translation Control

A family of protein kinases regulate translation initiation in response to cellular stresses by phosphorylation of eukaryotic initiation factor-2 (eIF-2). One family member from yeast, GCN2, contains a region homologous to histidyl-tRNA synthetases juxtaposed to the kinase catalytic domain. It is thought that uncharged tRNA accumulating during amino acid starvation binds to the synthetase-related sequences and stimulates phosphorylation of the alpha subunit of eIF-2. In this report, we define another domain in GCN2 that functions to target the kinase to ribosomes. A truncated version of GCN2 containing only amino acid residues 1467 to 1590 can independently associate with the translational machinery. Interestingly, this region of GCN2 shares sequence similarities with the core of the double-stranded RNA-binding domain (DRBD). Substitutions of the lysine residues conserved among DRBD sequences block association of GCN2 with ribosomes and impaired the ability of the kinase to stimulate translational control in response to amino acid limitation. Additionally, as found for other DRBD sequences, recombinant protein containing GCN2 residues 1467-1590 can bind double-stranded RNA in vitro, suggesting that interaction with rRNA mediates ribosome targeting. These results indicate that appropriate ribosome localization of the kinase is an obligate step in the mechanism leading to translational control by GCN2.

Effect of dietary methionine supplementation on hoof horn growth in primiparous cows

Horn growth rate does not appear to be related to the amino acid profile of the major protein source for dairy cattle (Offer, Logue &amp; Roberts, 1997), but it is possible that sulphur amino acids are limiting in early lactation, when the homeorhetic drive to milk production is most extreme. Supplementation of a high production ration for dairy cows with protected methionine should increase milk production, and allow any sulphur amino acid limitation on horn growth to be alleviated (Mengal, Galbraith, Souri &amp; Scaife, 1997).A total of 60 in-calf Holstein heifers were divided into two groups in a randomised block design. The study commenced approximately three weeks pre-calving until 26 weeks post calving, and animals were housed in one of three systems for the duration. Two diets were formulated based on a grass silage:maize silage mixture (50:50 DM basis), with rolled wheat, soya bean meal, sugar beet feed and rapeseed meal and were offered from approximately five days post calving for the remainder of the 26 week period.