The effects of maternal calcium status during late gestation on lamb growth and survival in twin-bearing Merino ewes grazing pasture.

Our project aimed to investigate whether low maternal calcium status during late gestation decreased weight, growth and survival of lambs born to twin-bearing Merino ewes grazing pasture. On day 130 of gestation, approximately a 10 mL blood sample was collected from 108 twin-bearing Merino ewes. Blood pH and calcium ion concentration were measured in whole blood and serum was analysed for calcium, magnesium, phosphate, beta hydroxy butyrate, non-esterified fatty acids and cholesterol. Six to 18 h after birth and at 49 days of age, lamb live weight was recorded. Ewe serum calcium level at day 130 of gestation were divided into tertiles (1.58-2.13, 2.14-2.30, and 2.31-2.99 mmol/L) with ewes classified as either Low, Med or High (n = 36 per group). A general linear mixed model was used to analyse the data. Survival to 1, 2, 3 and 7 days of age was lower for lambs born to ewes in the Low calcium group compared with those born in the Med and High calcium groups (P < 0.05). At day 49 of age, lamb survival tended to be lower for lambs in the Low (73.6%) compared with Med (86.8%) and High (85.3%) calcium groups (P < 0.1). There were no differences in lamb body weight (P > 0.05). Calcium levels of ewes in the Low calcium group were, on average, indicative of subclinical hypocalcaemia, with this group of ewes also having the lowest lamb survival rate, suggesting that maternal subclinical hypocalcaemia reduces lamb survival. Further studies, particularly on improving maternal calcium levels, are warranted.

Inhibition of Telomerase Activity Using an EGFP-Intron Splicing System Encoding Multiple RNAi Sequences.

To inhibit telomerase activity, a construct which contains artificial introns in the enhanced green fluorescent protein (EGFP) gene that encodes small hairpin RNA (shRNA) sequences that target human telomerase reverse transcriptase (hTERT) gene expression was designed and tested for its effect on lung cancer cell line. On intron splicing from the construct, intronic sequences were released and formed shRNA in the cells. After transfection of the construct, hTERT mRNA expression decreased by approximately 55 % in A549 cells. Correspondingly, in the same cell line, telomerase activity was decreased by approximately 23 %. The telomerase activity was transiently inhibited by this non-viral shRNA expression system that uses intron splicing to release artificial introns in an EGFP marker gene that contain shRNA targeting telomerase.

Potential roles for prions and protein-only inheritance in cancer.

Inherited mutations are known to cause familial cancers. However, the cause of sporadic cancers, which likely represent the majority of cancers, is yet to be elucidated. Sporadic cancers contain somatic mutations (including oncogenic mutations); however, the origin of these mutations is unclear. An intriguing possibility is that a stable alteration occurs in somatic cells prior to oncogenic mutations and promotes the subsequent accumulation of oncogenic mutations. This review explores the possible role of prions and protein-only inheritance in cancer. Genetic studies using lower eukaryotes, primarily yeast, have identified a large number of proteins as prions that confer dominant phenotypes with cytoplasmic (non-Mendelian) inheritance. Many of these have mammalian functional homologs. The human prion protein (PrP) is known to cause neurodegenerative diseases and has now been found to be upregulated in multiple cancers. PrP expression in cancer cells contributes to cancer progression and resistance to various cancer therapies. Epigenetic changes in the gene expression and hyperactivation of MAP kinase signaling, processes that in lower eukaryotes are affected by prions, play important roles in oncogenesis in humans. Prion phenomena in yeast appear to be influenced by stresses, and there is considerable evidence of the association of some amyloids with biologically positive functions. This suggests that if protein-only somatic inheritance exists in mammalian cells, it might contribute to cancer phenotypes. Here, we highlight evidence in the literature for an involvement of prion or prion-like mechanisms in cancer and how they may in the future be viewed as diagnostic markers and potential therapeutic targets.

Functions of Vrp1p in cytokinesis and actin patches are distinct and neither requires a WH2/V domain.

Vrp1 (verprolin, End5) is a Saccharomyces cerevisiae actin-associated protein and is related to mammalian Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP). Vrp1-deficient (vrp1 Delta) cells are inviable at high temperature, have partially depolarized cortical actin patches and have defects in both actomyosin ring-dependent and Hof1 (Cyk2)-dependent pathways of cytokinesis. We demonstrate here that N-Vrp1(1-364) and C-Vrp1(364-817) are each sufficient to restore viability, actomyosin ring constriction and Hof1 localization at 37 degrees C to vrp1 Delta. C-Vrp1, like Vrp1, partially co-localizes with cortical actin patches and restores actin patch polarization to vrp1 Delta. Cortical localization of C-Vrp1, but not Vrp1, requires Las17. N-Vrp1 exhibits diffuse cytoplasmic localization and functions in cytokinesis without efficiently restoring polarization of cortical actin patches. N-Vrp1 function is not abolished by mutations affecting the WASP homology 2 (WH2) [verprolin homology (V)] actin-binding domain. N-Vrp1 may function through the type I myosins and actin, while C-Vrp1 may function through both Las17 (Bee1) and type I myosins. The functions of Vrp1 in viability at 37 degrees C and cytokinesis do not require efficient localization to, and function in, the cortical actin cytoskeleton.

End13p/Vps4p is required for efficient transport from early to late endosomes in Saccharomyces cerevisiae.

end13-1 was isolated in a screen for endocytosis mutants and has been shown to have a post-internalisation defect in endocytic transport as well as a defect in vacuolar protein sorting (Vps(-) phenotype), leading to secretion of newly synthesised vacuolar proteins. Here we demonstrate that END13 is identical to VPS4, encoding an AAA (ATPase associated with a variety of cellular activities)-family ATPase. We also report that the end13-1 mutation is a serine 335 to phenylalanine substitution in the AAA-ATPase domain of End13p/Vps4p. It has been reported that mutant cells lacking End13p/Vps4p (end13(vps4)Delta) accumulate endocytosed marker dyes, plasma membrane receptors and newly synthesised vacuolar hydrolase precursors in an endosomal compartment adjacent to the vacuole (prevacuolar compartment, or PVC). We find, however, that the end13 mutants have defects in transport of endocytosed fluorescent dyes, plasma membrane receptors and ligands from small peripherally located early endosomes to larger late endosomes, which are often located adjacent to the vacuole. Our results indicate that End13p/Vps4p may play an important role in multiple steps of membrane traffic through the endocytic pathway.

Vrp1p functions in both actomyosin ring-dependent and Hof1p-dependent pathways of cytokinesis.

Vrp1p/verprolin/End5p is a Saccharomyces cerevisiae proline-rich protein, structurally and functionally related to human Wiskott-Aldrich syndrome protein-interacting protein. Vrp1p is required for viability at 37 degrees C, but not 24 degrees C. Here, we show that loss of Vrp1p (vrp1Delta) leads to a 3-4-fold delay in cytokinesis, wide bud necks, abnormal actomyosin rings, and aberrant septa even at 24 degrees C. Like other mutations affecting the actomyosin ring, vrp1Delta is synthetic lethal with deletion of HOF1 (or CYK2), which encodes a protein related to mammalian proline serine threonine phosphatase-interacting protein and Schizosaccharomyces pombe Cdc15p required for an actomyosin ring-independent pathway of cytokinesis in S. cerevisiae. At 37 degrees C, vrp1Delta cells rapidly cease dividing and exhibit a novel terminal phenotype: a single large bud, two well-separated nuclei, and an interphase microtubule array. The arrested cells have a persistent ring containing both actin and myosin at the bud neck. Many also exhibit some polarisation of cortical actin patches to the bud neck. Vrp1p binds an SH3-domain-containing fragment of Hof1p in vitro. Vrp1p is required in vivo for Hof1p relocalisation to a single ring at the bud neck prior to cytokinesis at 37 degrees C, but not at 24 degrees C. Vrp1p thus acts in both actomyosin ring formation and function, as well as in Hof1p localisation during cytokinesis.

Molecular requirements for the internalisation step of endocytosis: insights from yeast.

Molecular genetic studies of endocytosis using the unicellular eukaryote Saccharomyces cerevisiae (budding yeast) have led to the identification of many cellular components, both proteins and lipids, required for this process. While initially, many of these requirements (e.g. for actin, various actin-associated proteins, the ubiquitin conjugation system, and for ergosterol and sphingolipids) appeared to differ from known requirements for endocytosis in higher eukaryotes (e.g. clathrin, AP-2, dynamin), it now seems that endocytosis in higher and lower eukaryotes share many requirements. Often, what were initially identified as actin cytoskeleton-associated proteins in S. cerevisiae, are now revealing themselves as clathrin-coated pit- and vesicle-associated proteins in higher eukaryotes. So rather than delineating two endocytic pathways, one actin-based and one clathrin-based, the combined studies on higher and lower eukaryotes are revealing interesting interplay in both systems between the actin cytoskeleton, clathrin coats, and lipids in the formation of endocytic vesicles at the plasma membrane. Recent results from the yeast system show that the Arp2/3p complex, Wiskott-Aldrich syndrome protein (WASP), and WASP-interacting protein (WIP), proteins involved in the nucleation step of actin filament assembly, play a major role in the formation of endocytic vesicles. This discovery suggests models whereby endocytic vesicles may be actively pushed from the plasma membrane and into the cell by newly forming and rapidly extending actin filaments.

The yeast endocytic membrane transport system.

Progress has been made recently in visualizing the structures and organelles responsible for endocytic membrane traffic from the cell surface to the lysosome-like vacuole in Saccharomyces cerevisiae. This, together with the recent discovery of several new membrane trafficking pathways connecting these organelles, has led to a quantum leap in our understanding of the S. cerevisiae endocytic pathway. We now know that although the cortical actin cytoskeleton is required for the internalization step of endocytosis, the internalization event occurs at furrow-like invaginations of the plasma membrane, which are distinct from cortical actin patches. Internalized material is taken into the cell in the form of small (30-50 nm diameter) vesicles and delivered to tubulo-vesicular early endosomes at the cell periphery. Subsequently, the internalized material arrives in multivesicular late endosomes adjacent to the vacuole. Recent microscopy evidence suggests that transfer from late endosomes to the vacuole may involve direct fusion of late endosomes with the vacuole. The visualization of the S. cerevisiae endocytic pathway has revealed similarities to endocytic pathways visualized in higher eukaryotes.

Specific sterols required for the internalization step of endocytosis in yeast.

Sterols are major components of the plasma membrane, but their functions in this membrane are not well understood. We isolated a mutant defective in the internalization step of endocytosis in a gene (ERG2) encoding a C-8 sterol isomerase that acts in the late part of the ergosterol biosynthetic pathway. In the absence of Erg2p, yeast cells accumulate sterols structurally different from ergosterol, which is the major sterol in wild-type yeast. To investigate the structural requirements of ergosterol for endocytosis in more detail, several erg mutants (erg2Delta, erg6Delta, and erg2Deltaerg6Delta) were made. Analysis of fluid phase and receptor-mediated endocytosis indicates that changes in the sterol composition lead to a defect in the internalization step. Vesicle formation and fusion along the secretory pathway were not strongly affected in the ergDelta mutants. The severity of the endocytic defect correlates with changes in sterol structure and with the abundance of specific sterols in the ergDelta mutants. Desaturation of the B ring of the sterol molecules is important for the internalization step. A single desaturation at C-8,9 was not sufficient to support internalization at 37 degrees C whereas two double bonds, either at C-5,6 and C-7,8 or at C-5,6 and C-8,9, allowed internalization.

The WASp homologue Las17p functions with the WIP homologue End5p/verprolin and is essential for endocytosis in yeast.

Several end mutations that block the internalisation step of endocytosis in Saccharomyces cerevisiae also affect the cortical actin cytoskeleton [1]. END5 encodes a proline-rich protein (End5p or verprolin) required for a polarised cortical actin cytoskeleton and endocytosis [2,3]. End5p interacts with actin [4], but its exact function is not yet known. To help elucidate End5p function, we sought other End5p-interacting proteins and identified the LAS17/BEE1 gene (encoding the yeast homologue of the human Wiskott-Aldrich Syndrome protein, WASp) as a high-copy-number suppressor of the temperature-sensitive growth and endocytic defects of end5-1 cells (carrying a frameshift mutation affecting the last 213 residues of End5p). LAS17 is unable to suppress a full deletion of END5 (end5 delta), however, suggesting that the defective End5-1p in end5-1 mutants may be stabilised by Las17p. The amino terminus of Las17p interacts with the carboxyl terminus of End5p in the yeast two-hybrid system and similar interactions have been shown between WASp and a mammalian End5p homologue, WASp-interacting protein (WIP) [5]. As las17 delta deletion mutants are blocked in endocytosis, we conclude that Las17p and End5p interact and are essential for endocytosis.

End4p/Sla2p interacts with actin-associated proteins for endocytosis in Saccharomyces cerevisiae.

end4-1 was isolated as a temperature-sensitive endocytosis mutant. We cloned and sequenced END4 and found that it is identical to SLA2/MOP2. This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis. We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable. The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization. A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex. Furthermore, this domain has an endocytic function that is redundant with the function(s) of ABP1 and SRV2. The endocytic function of Abp1p depends on its SH3 domain. In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis. Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis.

Multiple classes of yeast mutants are defective in vacuole partitioning yet target vacuole proteins correctly.

In Saccharomyces cerevisiae the vacuoles are partitioned from mother cells to daughter cells in a cell-cycle-coordinated process. The molecular basis of this event remains obscure. To date, few yeast mutants had been identified that are defective in vacuole partitioning (vac), and most such mutants are also defective in vacuole protein sorting (vps) from the Golgi to the vacuole. Both the vps mutants and previously identified non-vps vac mutants display an altered vacuolar morphology. Here, we report a new method to monitor vacuole inheritance and the isolation of six new non-vps vac mutants. They define five complementation groups (VAC8-VAC12). Unlike mutants identified previously, three of the complementation groups exhibit normal vacuolar morphology. Zygote studies revealed that these vac mutants are also defective in intervacuole communication. Although at least four pathways of protein delivery to the vacuole are known, only the Vps pathway seems to significantly overlap with vacuole partitioning. Mutants defective in both vacuole partitioning and endocytosis or vacuole partitioning and autophagy were not observed. However, one of the new vac mutants was additionally defective in direct protein transport from the cytoplasm to the vacuole.

end5, end6, and end7: mutations that cause actin delocalization and block the internalization step of endocytosis in Saccharomyces cerevisiae.

Four mutants defective in endocytosis were isolated by screening a collection of temperature-sensitive yeast mutants. Three mutations define new END genes: end5-1, end6-1, and end7-1. The fourth mutation is in END4, a gene identified previously. The end5-1, end6-1, and end7-1 mutations do not affect vacuolar protein localization, indicating that the defect in each mutant is specific for internalization at the plasma membrane. Interestingly, localization of actin patches on the plasma membrane is affected in each of the mutants. end5-1, end6-1, and end7-1 are allelic to VRP1, RVS161, and ACT1, respectively. VRP1 and RVS161 are required for correct actin localization and ACT1 encodes actin. To our surprise, the end6-1 mutation fails to complement the act1-1 mutation. Disruption of the RVS167 gene, which is homologous to END6/RVS161 and which is also required for correct actin localization, also blocks endocytosis. The end7-1 mutant allele has a glycine 48 to aspartic acid substitution in the DNase I-binding loop of actin. We propose that Vrp1p, Rvs161p, and Rvs167p are components of a cytoskeletal structure that contains actin and fimbrin and that is required for formation of endocytic vesicles at the plasma membrane.

Endocytosis is required for the growth of vacuolar H(+)-ATPase-defective yeast: identification of six new END genes.

Yeast mutants that are defective in acidification of the lysosome-like vacuole are able to grow at pH 5.5, but not at pH 7. Here, we present evidence that endocytosis is required for this low pH-dependent growth and use this observation to develop a screen for mutants defective in endocytosis. By isolating mutants that cannot grow when they lack the 60-kD vacuolar ATPase subunit (encoded by the VAT2 gene), we isolated a number of vat2-synthetic lethal (Vsl-) mutant strains. Seven of the Vsl- mutants are defective in endocytosis. Four of these mutant strains (end8-1, end9-1, end10-1, and end11-1) show altered uptake of the endocytosed ligand, alpha-factor, and three (end12-1, end12-2, and end13-1) are probably defective in transfer of internalized material to the vacuole. Most of the mutations also confer a strong Ts- growth defect. The mutants defective in uptake of alpha-factor sort newly synthesized vacuolar proteins correctly, while those which may be defective in subsequent transport steps secrete at least a fraction of the newly synthesized soluble vacuolar proteins. The mutations that result in a defect in alpha-factor uptake are not allelic to any of the genes previously shown to encode endocytic functions.

The chloroplast beta-subunit allows assembly of the Escherichia coli F0 portion of the energy transducing adenosine triphosphatase.

The effect of the expression of the chloroplast F1-ATPase beta-subunit in two Escherichia coli beta-subunit mutant strains was investigated. The amount of chloroplast beta-subunit formed in E. coli was increased by introducing a 'Shine-Dalgarno' sequence upstream from the translation start site. The chloroplast beta-subunit was membrane bound but was unable to functionally replace the mutant beta-subunit in a strain carrying the uncD409 allele [corrected]. However, in an E. coli mutant strain unable to form the beta- and epsilon-subunits the presence of the chloroplast beta-subunit enabled the assembly of a functional proton pore [corrected]

Viability of clathrin heavy-chain-deficient Saccharomyces cerevisiae is compromised by mutations at numerous loci: implications for the suppression hypothesis.

The gene encoding clathrin heavy chain in Saccharomyces cerevisiae (CHC1) is not essential for growth in most laboratory strains tested. However, in certain genetic backgrounds, a deletion of CHC1 (chc1) results in cell death. Lethality in these chc1 strains is determined by a locus designated SCD1 (suppressor of clathrin deficiency) which is unlinked to CHC1 (S. K. Lemmon and E. W. Jones, Science 238:504-509, 1987). The lethal allele of SCD1 has no effect on cell growth when the wild-type version of CHC1 is present. This result led to the proposal that most yeast strains are viable in the absence of clathrin heavy chain because they possess the SCD1 suppressor. Discovery of another yeast strain that cannot grow without clathrin heavy chain has allowed us to perform a genetic test of the suppressor hypothesis. Genetic crosses show that clathrin-deficient lethality in the latter strain is conferred by a single genetic locus (termed CDL1, for clathrin-deficient lethality). By constructing strains in which CHC1 expression is regulated by the GAL10 promoter, we demonstrate that the lethal alleles of SCD1 and CDL1 are recessive. In both cases, very low expression of CHC1 can allow cells to escape from lethality. Genetic complementation and segregation analyses indicate that CDL1 and SCD1 are distinct genes. The lethal CDL1 allele does not cause a defect in the secretory pathway of either wild-type or clathrin heavy-chain-deficient yeast. A systematic screen to identify mutants unable to grow in the absence of clathrin heavy chain uncovered numerous genes similar to SCD1 and CDL1. These findings argue against the idea that viability of chc1 cells is due to genetic suppression, since this hypothesis would require the existence of a large number of unlinked genes, all of which are required for suppression. Instead, lethality appears to be a common, nonspecific occurrence when a second-site mutation arises in a strain whose cell growth is already severely compromised by the lack of clathrin heavy chain.

The chloroplast CF0I subunit can replace the b-subunit of the F0F1-ATPase in a mutant strain of Escherichia coli K12.

The amino acid sequence of the CF0I subunit from the chloroplast F0F1-ATPase has only a low similarity to the amino acid sequence of the b-subunit of the E. coli F0F1-ATPase. However, secondary and tertiary structure predictions plus the distribution of hydrophobic and hydrophilic amino acids have indicated that these two subunits serve a similar function. This proposition was investigated directly. A cDNA clone for the chloroplast atpF gene, encoding the CF0I subunit, was altered by site-directed mutagensis such that the translation start site corresponded to the N-terminus of the mature protein. An E. coli mutant strain carrying a chain-terminating mutation in the uncF gene, encoding the b-subunit, was transformed with the plasmid carrying the altered atpF gene. The resultant transformant was able to grow on succinate and gave a growth yield similar to that of a wild-type control. Assays on membrane preparations from the transformant also clearly indicated that the mature CF0I subunit from spinach chloroplasts was able to replace the E. coli b-subunit in the E. coli F0F1-ATPase.

High-level synthesis of the phage lambda outer-membrane protein from the cloned lom gene.

A 2.7-kb KpnI-EcoRI fragment carrying the lom gene of bacteriophage lambda has been cloned into plasmid pPR42 and recloned into the SmaI site of pUC9. Large quantities of Lom were seen in outer-membrane (OM) preparations of strains carrying the latter clone and its derivatives. The reading frame of lom was identified as ORF206a. The protein was not demonstrably associated either covalently or non-covalently with the peptidoglycan layer of the cell envelope.