Comparison of two commercial extenders for cryopreservation of goat semen without sperm washing.

The objective of this study was to evaluate the effects of two commercially available semen extenders on the motility of cryopreserved goat sperm and to simplify the cryopreservation protocol. Individual goat ejaculates were split and processed in parallel for freezing in either commercially available soy-based extender (Bioxcell®) or egg yolk-based extender (Irvine TYB). Sperm quality was assessed using total and progressive sperm motility, measured by computer-assisted sperm analysis (CASA). Total motility was higher for samples processed in soy-based extender, both at pre-freeze (P = 0.002) and at post-thaw (P < 0.0001). Progressive motility was higher for semen processed in soy extender at post-thaw (P < 0.0001). Approximately 10% of samples processed in egg yolk-based extender had a large (> 50%) reduction in total motility prior to freezing. However, this type of extreme reduction in pre-freeze motility did not occur in semen samples processed in soy extender. In addition, the use of soy-based extender eliminated the need for a time-consuming sperm washing protocol. We concluded that a commercially available soy-based extender was superior to an egg yolk-based extender in preserving motility of cryopreserved goat sperm, using a two-step method.

Normal retinal development and retinofugal projections in mice lacking the retina-specific variant of actin-binding LIM domain protein.

The actin-binding LIM domain protein (abLIM) is the mammalian homologue of UNC-115, a protein mediating axon guidance in C. elegans. AbLIM is widely expressed with three isoforms differing from one another by the length of their amino termini. Experiments utilizing dominant-negative mutants in the chick retina suggested a role for abLIM in axon path finding in retinal ganglion cells (RGCs). To investigate which variant is involved in the regulation of mammalian RGC axon guidance, we analyzed their expression profile in mice. The longest variant, abLIM-L, is highly enriched in the ganglion cell layer. AbLIM-L is up-regulated postnatally which temporally overlaps with the period of RGC axon remodeling. In contrast, the abLIM-M and abLIM-S variants are widespread and remain relatively constant through development. By selective gene targeting, we ablated abLIM-L to explore its functional significance in vivo. AbLIM-L mutant mice exhibit no apparent morphological or functional defects in photoreceptors and inner retinal neurons. Retinofugal projections and synaptic maturation also appear normal. These data suggest that abLIM-M is likely the isoform performing the essential function related to axon guidance.

Degradation of the kinesin Kip1p at anaphase onset is mediated by the anaphase-promoting complex and Cdc20p.

Kip1p of Saccharomyces cerevisiae is a bipolar kinesin in the conserved bimC kinesin subfamily that mediates mitotic spindle-pole separation. Here, we show that Kip1p is regulated immediately after anaphase initiation by its rapid degradation. Degradation required the ubiquitin protein ligase called the anaphase-promoting complex, the anaphase-promoting complex activating protein Cdc20, and a unique 43-aa sequence in Kip1p. Degradation also required import of Kip1p into the nucleus, but occurred independently of spindle association. A mutation that stabilized Kip1p impaired anaphase progression. The timing of degradation suggests that Kip1p functions primarily during spindle assembly and metaphase, and that Kip1p degradation facilitates structural changes in the mitotic spindle as anaphase progresses.

Saccharomyces cerevisiae Mob1p is required for cytokinesis and mitotic exit.

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G(1) by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1 mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize in mob1 mutants, suggesting that MOB1 functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck required CDC3, MEN genes CDC5, CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent of MYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14 mutants. These results suggest that the MEN functions during the mitosis-to-G(1) transition to control cyclin-CDK inactivation and cytokinesis.

The kinesin-related protein Kip1p of Saccharomyces cerevisiae is bipolar.

Kip1p is a mitotic spindle-associated kinesin-related protein in Saccharomyces cerevisiae that participates in spindle pole separation. Here, we define the domain arrangement and polypeptide composition of the Kip1p holoenzyme. Electron microscopy of rotary shadowed Kip1p molecules revealed two globular domains 14 nm in diameter connected by a 73-nm long stalk. When the Kip1p domain homologous to the kinesin motor domain was decorated with an unrelated protein, the diameter of the globular domains at both ends of the stalk increased, indicating that Kip1p is bipolar. Soluble Kip1p isolated from S. cerevisiae cells was homomeric, based on the similarity of the sedimentation coefficients of native Kip1p from S. cerevisiae and Kip1p which was purified after expression in insect cells. The holoenzyme molecular weight was estimated using the sedimentation coefficient and Stokes radius, and was most consistent with a tetrameric composition. Kip1p exhibited an ionic strength-dependent transition in its sedimentation coefficient, revealing a potential regulatory mechanism. The position of kinesin motor-related domains at each end of the stalk may allow Kip1p to cross-link either parallel or antiparallel microtubules during mitotic spindle assembly and pole separation.

Kinesin-related KIP3 of Saccharomyces cerevisiae is required for a distinct step in nuclear migration.

Spindle orientation and nuclear migration are crucial events in cell growth and differentiation of many eukaryotes. Here we show that KIP3, the sixth and final kinesin-related gene in Saccharomyces cerevisiae, is required for migration of the nucleus to the bud site in preparation for mitosis. The position of the nucleus in the cell and the orientation of the mitotic spindle was examined by microscopy of fixed cells and by time-lapse microscopy of individual live cells. Mutations in KIP3 and in the dynein heavy chain gene defined two distinct phases of nuclear migration: a KIP3-dependent movement of the nucleus toward the incipient bud site and a dynein-dependent translocation of the nucleus through the bud neck during anaphase. Loss of KIP3 function disrupts the unidirectional movement of the nucleus toward the bud and mitotic spindle orientation, causing large oscillations in nuclear position. The oscillatory motions sometimes brought the nucleus in close proximity to the bud neck, possibly accounting for the viability of a kip3 null mutant. The kip3 null mutant exhibits normal translocation of the nucleus through the neck and normal spindle pole separation kinetics during anaphase. Simultaneous loss of KIP3 and kinesin-related KAR3 function, or of KIP3 and dynein function, is lethal but does not block any additional detectable movement. This suggests that the lethality is due to the combination of sequential and possibly overlapping defects. Epitope-tagged Kip3p localizes to astral and central spindle microtubules and is also present throughout the cytoplasm and nucleus.

Respiratory deficiency due to loss of mitochondrial DNA in yeast lacking the frataxin homologue.

Friedreich's ataxia (FRDA) is an autosomal recessive degenerative disorder that primarily affects the nervous system and heart. Patients with FRDA have point mutations or trinucleotide repeat expansions in both alleles of FRDA, which encodes a protein termed frataxin. We show that the yeast frataxin homologue, which we have named YFH1, localizes to mitochondria and is required to maintain mitochondrial DNA. The YFH1-homologous domain of frataxin functions in yeast and a disease-associated missense mutation of this domain, or the corresponding domain in YFH1, reduces function. Our data suggest that mitochondrial dysfunction contributes to FRDA pathophysiology.

Molecular characterization of abLIM, a novel actin-binding and double zinc finger protein.

Molecules that couple the actin-based cytoskeleton to intracellular signaling pathways are central to the processes of cellular morphogenesis and differentiation. We have characterized a novel protein, the actin-binding LIM (abLIM) protein, which could mediate such interactions between actin filaments and cytoplasmic targets. abLIM protein consists of a COOH-terminal cytoskeletal domain that is fused to an NH2-terminal domain consisting of four double zinc finger motifs. The cytoskeletal domain is approximately 50% identical to erythrocyte dematin, an actin-bundling protein of the red cell membrane skeleton, while the zinc finger domains conform to the LIM motif consensus sequence. In vitro expression studies demonstrate that abLIM protein can bind to F-actin through the dematin-like domain. Transcripts corresponding to three distinct isoforms have a widespread tissue distribution. However, a polypeptide corresponding to the full-length isoform is found exclusively in the retina and is enriched in biochemical extracts of retinal rod inner segments. abLIM protein also undergoes extensive phosphorylation in light-adapted retinas in vivo, and its developmental expression in the retina coincides with the elaboration of photoreceptor inner and outer segments. Based on the composite primary structure of abLIM protein, actin-binding capacity, potential regulation via phosphorylation, and isoform expression pattern, we speculate that abLIM may play a general role in bridging the actin-based cytoskeleton with an array of potential LIM protein-binding partners. The developmental time course of abLIM expression in the retina suggests that the retina-specific isoform may have a specialized role in the development or elaboration of photoreceptor inner and outer segments.

Rhodopsin accumulation at abnormal sites in retinas of mice with a human P23H rhodopsin transgene.

PURPOSE: To investigate the mechanism by which photoreceptors degenerate in transgenic mice carrying a mutant human rhodopsin gene (P23H). METHODS: The temporal and spatial pattern of the retinal degeneration caused by P23H rhodopsin was mapped using immunocytochemistry with rhodopsin-specific antibodies. The subcellular localizations of rhodopsin, transducin, and rod cGMP phosphodiesterase (PDE) were also determined, and rhodopsin localization was compared among P23H transgenic mice, rd mice, and Royal College of Surgeons (RCS) rats. RESULTS: In transgenic mice that express P23H rhodopsin, photoreceptors are lost centrally by postnatal day 10. As the retina degenerates, rhodopsin accumulates in the outer nuclear layer and within the photoreceptor synaptic terminals. The P23H transgenic retinas also show an accumulation of transducin and PDE within the outer plexiform layer. In contrast, other types of hereditary retinal degenerations studied show a similar pattern of rhodopsin accumulation in the outer nuclear layer but not in the outer plexiform layer of the retina. CONCLUSIONS: The pattern of degeneration in the P23H transgenic retina is consistent with a model in which the centrally located, first-born photoreceptors are the first to die. In contrast to other animal models for hereditary retinal degeneration (rd, RCS), a novel feature of the P23H degeneration is an accumulation of rhodopsin, transducin, and PDE within the outer plexiform layer of the retina. One hypothesis to explain this observation is that P23H rhodopsin is routed intracellularly through a pathway not used by normal rhodopsin. Nonmutant forms of the peripheral transducing proteins normally associated with disk membrane, such as transducin and PDE, may accompany the aberrantly routed rhodopsin.

In vivo transfer of a reporter gene to the retina mediated by an adenoviral vector.

PURPOSE: The ability of replication-deficient adenovirus to mediate gene transfer to retinal cells was evaluated. METHODS: A replication-deficient adenoviral vector, AdCMV beta A.ntlacZ, which contains the bacterial beta-galactosidase (lacZ) reporter gene, was injected into the subretinal space of normal, rd, and rds strains of mice at various ages. The efficiency and duration of transgene expression were assessed by histochemical examination and transmission electron microscopy. RESULTS: AdCMV beta A.ntlacZ was effective in mediating gene transfer to the retinal pigment epithelial cells, rod and cone photoreceptor cells, and cells in the inner nuclear layer of the retina for periods of up to 1 month. Gene transfer to retinal pigment epithelial cells occurred at much lower viral titers than was required for gene transfer to photoreceptor cells. The extent to which photoreceptor cells could be transduced varied with the age of the animals and the conditions of the photoreceptor cells: greater numbers of photoreceptor cells were transduced in 5- to 7-day-old pups and in mice at the initial stages of photoreceptor degeneration than in normal adult mice. No evidence of gross pathogenic effects or viremia in recipient mice was observed. CONCLUSIONS: Replication-deficient adenovirus mediates transfer and expression of a foreign gene in retinal pigment epithelial and photoreceptor cells. Gene transfer to photoreceptor cells is enhanced in developing retinas or at the predegenerate stage of photoreceptors in genetically programmed retinal degeneration.

Transgenic mice with a rhodopsin mutation (Pro23His): a mouse model of autosomal dominant retinitis pigmentosa.

We inserted into the germline of mice either a mutant or wild-type allele from a patient with retinitis pigmentosa and a missense mutation (P23H) in the rhodopsin gene. All three lines of transgenic mice with the mutant allele developed photoreceptor degeneration; the one with the least severe retinal photoreceptor degeneration had the lowest transgene expression, which was one-sixth the level of endogenous murine rod opsin. Of two lines of mice with the wild-type allele, one expressed approximately equal amounts of transgenic and murine opsin and maintained normal retinal function and structure. The other expressed approximately 5 times more transgenic than murine opsin and developed a retinal degeneration similar to that found in mice carrying a mutant allele, presumably due to the overexpression of this protein. Our findings help to establish the pathogenicity of mutant human P23H rod opsin and suggest that overexpression of wild-type human rod opsin leads to a remarkably similar photoreceptor degeneration.

Autogenous regulation of ethanolamine utilization by a transcriptional activator of the eut operon in Salmonella typhimurium.

The genes required for use of ethanolamine as a carbon and nitrogen source are encoded by a single operon (eut) whose expression is induced by the simultaneous presence of both ethanolamine and cobalamin (vitamin B12). The action of B12 as an inducer of this operon reflects the fact that this cofactor is required by the degradative enzyme ethanolamine lyase (eutBC). The eutR gene encodes a protein that activates transcription of the eut operon in response to the simultaneous presence of B12 and ethanolamine. The eutR gene is expressed by a weak constitutive promoter activity (PII) and by the main regulated promoter (PI). Because it is encoded within the operon that it activates, the EutR protein controls its own production. Initial induction of the eut operon by ethanolamine plus B12 causes an increase in expression of the eutR gene; this increase acts as part of a positive feedback loop that is required for maximal operon expression. Because of this mode of regulation, constitutive regulatory mutations, described here, include mutations that generate new internal promoters and thereby increase the basal level of eutR gene expression. In mutants with an increased level of activator protein, each inducer (B12 or ethanolamine), presented singly, is sufficient for partial operon induction.

Kinesin-related proteins required for assembly of the mitotic spindle.

We identified two new Saccharomyces cerevisiae kinesin-related genes, KIP1 and KIP2, using polymerase chain reaction primers corresponding to highly conserved regions of the kinesin motor domain. Both KIP proteins are expressed in vivo, but deletion mutations conferred no phenotype. Moreover, kip1 kip2 double mutants and a triple mutant with kinesin-related kar3 had no synthetic phenotype. Using a genetic screen for mutations that make KIP1 essential, we identified another gene, KSL2, which proved to be another kinesin-related gene, CIN8. KIP1 and CIN8 are functionally redundant: double mutants arrested in mitosis whereas the single mutants did not. The microtubule organizing centers of arrested cells were duplicated but unseparated, indicating that KIP1 or CIN8 is required for mitotic spindle assembly. Consistent with this role, KIP1 protein was found to colocalize with the mitotic spindle.

Cytoskeletal specializations at the rod photoreceptor distal tip.

We have examined microtubules and microtubule-like elements within the toad rod photoreceptor outer segment in order to define regional specializations of the photoreceptor cytoskeleton. "Ciliary" microtubules were localized within the rod outer segment (ROS) by using thin section electron microscopy, immunofluorescence, and rapid-freeze deep-etch microscopy. All three methods showed that ciliary microtubules stop short of the extreme ROS distal tip, although abundant microtubule-like structures distinct from the ciliary microtubules were found within the distal 10-15 microns of the ROS tip. These heretofore undescribed "distal ROS tubules" are clustered at the clefts or incisures of the disk membrane stack and resemble microtubules in overall size and shape, although they are not closely related antigenically to tubulin. The distal ROS tubules are more abundant in green rods than red rods and vary in number during the daily light/dark cycle. Quantitation of these tubules at two time points during the light/dark cycle suggests that there are three- to fourfold more tubules in the ROS tip one hour after light onset than one hour before light onset. Retinas prevented from normal disk membrane shedding by separation of the retina from the adjacent pigment epithelium, failed to develop increased numbers of tubules after light onset. This suggests that the newly described distal ROS tubules may modulate or be modulated by light-induced interactions between the photoreceptors and pigment epithelium, such as those that occur during the disk shedding phase of membrane turnover.

A 52 kD cytoskeletal protein from retinal rod photoreceptors is related to erythrocyte dematin.

A novel cytoskeletal antigen, RET52, has been identified in the mouse retina. This 52 kD polypeptide is antigenically related to dematin (band 4.9), an actin-bundling phosphoprotein component of the erythrocyte membrane skeleton. Like dematin, RET52 is also a substrate for cAMP-dependent protein kinase. Within the retina, RET52 is primarily concentrated in two regions-the rod inner segment and the outer synaptic layers-although the developmental expression of RET52 differs in these areas. RET52 is present at birth in the inner segment, but appears about the time of initial synapse formation (postnatal day 4-6) in the outer plexiform layer. No differences in RET52 expression have been detected in early-stage mouse retinas with the retinal degeneration (rd) phenotype. RET52 localization, developmental expression, homologies to dematin, and in vitro phosphorylation pattern suggest a possible role for cytoskeleton-associated proteins in the initiation or control of disk membrane assembly and/or synapse formation in the rod photoreceptor.

Cloning, sequencing, and expression of the genes encoding the adenosylcobalamin-dependent ethanolamine ammonia-lyase of Salmonella typhimurium.

Ethanolamine ammonia-lyase is a bacterial enzyme that catalyzes the adenosylcobalamin-dependent conversion of certain vicinal amino alcohols to oxo compounds and ammonia. Studies of ethanolamine ammonia-lyase from Clostridium sp. and Escherichia coli have suggested that the enzyme is a heterodimer composed of subunits of Mr approximately 55,000 and 35,000. Using a partial Sau3A Salmonella typhimurium library ligated into pBR328 and selecting by complementation of a mutant lacking ethanolamine ammonia-lyase activity, we have cloned the genes for the 2 subunits of the S. typhimurium enzyme. The genes were localized to a 6.5-kilobase fragment of S. typhimurium DNA, from which they could be expressed in E. coli under noninducing conditions. Sequencing of a 2526-base pair portion of this 6.5-kilobase DNA fragment revealed two open reading frames separated by 21 base pairs. The open reading frames encoded proteins of 452 and 286 residues whose derived N-terminal sequences were identical to the N-terminal sequences of the 2 subunits of the E. coli ethanolamine ammonia-lyase, except that residue 16 of the large subunit was asparagine in the E. coli sequence and aspartic acid in the S. typhimurium sequence.

Functions required for vitamin B12-dependent ethanolamine utilization in Salmonella typhimurium.

When B12 is available, Salmonella typhimurium can degrade ethanolamine to provide a source of carbon and nitrogen. B12 is essential since it is a cofactor for ethanolamine ammonia-lyase, the first enzyme in ethanolamine breakdown. S. typhimurium makes B12 only under anaerobic conditions; in the presence of oxygen, exogenous B12 must be provided to permit ethanolamine utilization. Genes required for ethanolamine utilization are encoded in the eut operon. For complementation testing, an F' plasmid containing the eut genes was constructed by transposition of the eut operon (flanked by two Tn10 elements) to an existing F plasmid. Complementation tests defined six genes in the eut operon. Three of these genes encode enzymes known to be involved in degradation of ethanolamine: ethanolamine ammonia-lyase (eutB and eutC) and acetaldehyde dehydrogenase (eutE). One gene (eutR) seems to encode a positive regulatory protein required for induction of transcription of eut. The function of one of the remaining two genes (eutA) was shown to be required for ethanolamine utilization only when cyano-B12 or hydroxy-B12 were the precursors of the adenosyl-B12 cofactor of ethanolamine ammonia-lyase; eutA mutants could use ethanolamine as the nitrogen source only when adenosyl-B12 was provided. No function has been assigned to the eutD gene, which is required for use of ethanolamine as a carbon source. Ethanolamine uptake assays of eut mutants suggest that no ethanolamine permease is encoded in the eut operon.

Ethanolamine utilization in Salmonella typhimurium.

Ethanolamine can serve as the sole source of carbon and nitrogen for Salmonella typhimurium if vitamin B12 is present to serve as a cofactor. The pathway for ethanolamine utilization has been investigated in order to understand its regulation and determine whether the pathway is important to the selective forces that have maintained the ability to synthesize B12 in S. typhimurium. We isolated mutants that are defective in ethanolamine utilization (eut mutants). These mutants defined a cluster of genes located between purC and cysA at 50 min on the Salmonella chromosome. A genetic map of the eut region was constructed. Included in the map are mutations which affect ethanolamine ammonia lyase, the first degradative enzyme, and mutations which affect the second enzyme in the pathway, acetaldehyde dehydrogenase. Transcriptional regulation of the eut genes was studied by using eut-lac operon fusions created by insertion of Mu d lac. Transcription is induced by the simultaneous presence of ethanolamine and B12 in the growth medium. The eut genes constitute a single unit of transcription. One class of mutations located at the promoter-distal end of the eut operon prevent induction of transcription.