A mouse translocation associated with Caspr5-2 disruption and perinatal lethality

We have previously described the paralogous mouse genes Caspr5-1, -2, and -3 of the neurexin gene family. Here we present the cytogenetic and molecular mapping of a null mutation of Caspr5-2 which was caused by reciprocal translocation between chromosomes 1 and 8 with breakpoints at bands 1E2.1 and 8B2.1, respectively. The translocation disrupts Caspr5-2 between exons 1 and 2 and causes stillbirth or early postnatal lethality of homozygous carriers. Because no other candidate genes were found, the disruption of Caspr5-2 is most likely the cause of lethality. Only rarely do homozygotes survive the critical stage, reach fertility, and are then apparently normal. They may be rescued by one of the two other Caspr5 paralogs. Caspr5-2 is expressed in spinal cord and brain tissues. Despite giving special attention to regions where in wild-type fetuses maximum expression was found, no malformation that might have caused death could be detected in fetal homozygous carriers of the translocation. We, therefore, suspect that Caspr5-2 disruption leads to dysfunction at the cellular level rather than at the level of organ development.     

The inter- and intrachromosomal effects of the bar-stone translocation in Drosophila melanogaster

Summary Females homozygous and heterozygous for the B ^S translocation were tested to determine the extent of the intra- and interchromosomal effects caused by the rearrangement. The heterozygous translocation produces an increase in crossing over at the tip of the X and in the centromere region of chromosome 2. The homozygous translocation has no effect on crossing over in these regions, but an unexpected increase is observed near the centromere region of the B ^S segment. This result is not predicted by the time-delay model for interchromsomal effects.Supported by USPHS Training Grant 5T1 GM 1145-05 and NSF GB 18786.     

Seedling lethality in wheat: a novel phenotype associated with a 2RS/2BL translocation chromosome

Summary When grown at normal temperatures, wheat plants disomic for a 2RS/2BL translocation chromosome substituting for chromosome 2B show seedling lethality. Morphological and physiological studies could not determine the cause of death. However, the seedling lethality can be partly to completely inactivated at higher temperatures and in stressed environments. The lethality can also be completely suppressed if the translocated chromosome is introduced into different wheat cultivars. These wheats must contain genes which suppress the lethal phenotype caused by disomy of 2RS/2BL.Whilst the temperature effect indicates that the seedling lethality is related to the grass clump dwarf phenotype of wheat, our results show that the genes involved in seedling lethality, its suppression and inactivation, are not related to the D genes which cause grass clump dwarfing in wheat.     

Mutations in CDC14 result in high sensitivity to cyclin gene dosage in Saccharomyces cerevisiae

We screened for mutations that resulted in lethality when the G1 cyclin Cln2p was overexpressed throughout the cell cycle in Saccharomyces cerevisiae. Mutations in five complementation groups were found to give this phenotype, and three of the mutated genes were identified as MEC1, NUP170, and CDC14. Mutations in CDC14 may have been recovered in the screen because Cdc14p may reduce the cyclin B (Clb)-associated Cdc28 kinase activity in late mitosis, and Cln2p may normally activate Clb-Cdc28 kinase activity by related mechanisms. In agreement with the idea that cdc14 mutations elevate Clb-Cdc28 kinase activity, deletion of the gene for the Clb-Cdc28 inhibitor Sic1 caused synthetic lethality with cdc14-1, as did the deletion of HCT1, which is required for proteolysis of Clb2p. Surprisingly, deletion of the gene for the major B-type cyclin, CLB2, also caused synthetic lethality with the cdc14-1 mutation. The clb2 cdc14 strains arrested with replicated but unseparated DNA and unseparated spindle pole bodies; this phenotype is distinct from the late mitotic arrest of the sic1::TRP1 cdc14-1 and the cdc14-1 hct1::LEU2 double mutants and of the cdc14 CLN2 overexpressor. We found genetic interactions between CDC14 and the replication initiator gene CDC6, extending previous observations of interactions between the late mitotic function of Cdc14p and control of DNA replication. We also describe genetic interactions between CDC28 and CDC14. Received: 24 May 1999 / Accepted: 19 October 1999     

Haplotypic variation of the transporter associated with antigen processing (TAP) genes and their extension of HLA class II region haplotypes

Stable cell surface presentation of HLA class I molecules requires active transport of antigenic peptides across the endoplasmic reticulum by products of two genes, TAP1 and TAP2, which map in the major histocompatibility complex class II region. Alleles of each gene are derived from a combination of variable sitesaat each locus. In this study, TAP1 and TAP2 alleles were identified in homozygous typing cell (HTC) lines, allowing resolution of specific haplotypes in conjunction with the highly polymorphic HLA class II region haplotypes. Three alleles at each TAP locus were found from which eight haplotypes could be assigned. Determination of TAP1 and TAP2 alleles in cell lines homozygous at DR, DQ, and DP created eight additional haplotypes beyond the number observed with these class II genes alone. Complete analysis of DR, DQ, TAP, and DP genotypes in 66 HTCs resulted in the following groups: 1) 46 homozygotes; 2) nine homozygous at DR, DQ, and TAP, but heterozygous at DP; 3) four homozygous at DR, DQ, and DP, but heterozygous at one or both TAP genes; 4) four homozygous at DR and DQ, but heterozygous at TAP and DP; and 5) three complex genotypes heterozygous at DP, TAP, and at least one of DQA1, DQB1, or DRB1 loci. TAP1 and TAP2 genes map in an area of frequent recombination. TAP alleles were determined in five DQB1, DPB1 recombinant individuals, three of which were informative. Recombination was found between DQB1 and the TAP loci in two individuals and between TAP and DPB1 in the other individual.     

CDK-1 and Two B-Type Cyclins Promote PAR-6 Stabilization during Polarization of the Early C. elegans Embryo

In the C. elegans embryo, formation of an antero-posterior axis of polarity relies on signaling by the conserved PAR proteins, which localize asymmetrically in two mutually exclusive groups at the embryonic cortex. Depletion of any PAR protein causes a loss of polarity and embryonic lethality. A genome-wide RNAi screen previously identified two B-type cyclins, cyb-2.1 and cyb-2.2, as suppressors of par-2(it5ts) lethality. We found that the loss of cyb-2.1 or cyb-2.2 suppressed the lethality and polarity defects of par-2(it5ts) mutants and that these cyclins act in cell polarity with their cyclin-dependent kinase partner, CDK-1. Interestingly, cyb-2.1; cyb-2.2 double mutants did not show defects in cell cycle progression or timing of polarity establishment, suggesting that they regulate polarity independently of their typical role in cell cycle progression. Loss of both cyclin genes or of cdk-1 resulted in a decrease in PAR-6 levels in the embryo. Furthermore, the activity of the cullin CUL-2 was required to achieve suppression of par-2 lethality when both cyclins were absent. Our results support a model in which CYB-2.1/2/CDK-1 antagonize CUL-2 activity to promote stabilization of PAR-6 levels during polarization of the early C. elegans embryo. They also suggest that CYB-2.1 and CYB-2.2 contribute to the coupling of cell cycle progression and asymmetric segregation of cell fate determinants.     

Towards a male-only release system for SIT with the Queensland fruit fly,Bactrocera tryoni,using a genetic sexing strain with a temperature-sensitive lethal mutation

Flies that are homozygous for the recessive autosomal mutation bent wingshave a limited ability to fly and are less tolerant of high temperatures than normal flies in both the egg and puparial stages. The differences between the mutant and normal flies were found sufficient to be the basis of a genetic sexing strain. Genetic sexing strains were created using translocations of the autosome bearing the wild-type allele of bent wings(chromosome 2) to the Y chromosome, and crossing male flies carrying the translocation to mutant bent wingsfemales. In the resulting strain, the females were homozygous for the bent wingsmutation and the males were phenotypically normal for wing characters. Several translocations were recovered after irradiation, but only one translocation involving chromosome 2 was both stable and expressed in a stock that was vigorous enough for long-term viability. Unfortunately, all stocks containing the translocation showed high levels of temperature-dependent lethality, including, inexplicably, both males and females. Translocation stocks showing this effect included bent wings, another second chromosome mutation, white marks, and an otherwise normal stock. This phenomenon is probably rare, as it has not been reported before. It is likely that bent wingscould be suitably used with another translocation.     

Strategies for Pyramiding Resistance Genes Against the Barley Yellow Mosaic Virus Complex (BaMMV,BaYMV, BaYMV-2)

Barley Yellow Mosaic Virus disease caused by different strains of BaYMV and BaMMV is a major threat to winter barley cultivation in Europe. Pyramiding of resistance genes may be considered as a promising strategy to avoid the selection of new virus strains and to create more durable resistances. However, this goal cannot be achieved by phenotypic selection due to the lack of differentiating virus strains. For pyramiding of resistance genes rym4, rym5, rym9 and rym11, located on chromosomes 3H and 4H of barley two different strategies have been developed. These strategies are based on doubled haploid lines (DHs) and marker assisted selection procedures. On the one hand F₁ derived DH-plants of single crosses were screened by molecular markers for genotypes being homozygous recessive for both resistance genes. These genotypes were crossed to lines carrying one resistance gene in common and an additional third gene, leading to a DH-population of which 25% carry three resistance genes, 50% have two resistance genes and 25% possess a single resistance gene homozygous recessively. Alternatively, F₁ plants having one resistance gene in common were directly inter-crossed [e.g. (rym4 × rym9) × (rym4 × rym11)] and about 100 seeds were produced per combination. Within these complex cross progenies plants were identified by markers being homozygous at the common resistance locus and heterozygous at the others. From such plants, theoretically present at a frequency of 6.25%, DH-lines were produced, which were screened for the presence of genotypes carrying three or two recessive resistance genes in a homozygous state. Besides DH-plants carrying all possible two-gene combinations, 20 DH-plants out of 107 analysed carrying rym4, rym9, and rym11 and 27 out of 187 tested carrying rym5, rym9, and rym11 homozygously have been detected using the second strategy which is faster but needs co-dominant markers, because in contrast to the first strategy marker selection is carried out on heterozygous genotypes.     

New members of the neurexin superfamily: multiple rodent homologues of the human CASPR5 gene

Proteins of the Caspr family are involved in cell contacts and communication in the nervous system. We identified and, by in silico reconstruction, compiled three orthologues of the human CASPR5 gene from the mouse genome, four from the rat genome, and one each from the chimpanzee, dog, opossum, and chicken genomes. Obviously, Caspr5 gene duplications have taken place during evolution of the rodent lineage. In the rat, the four paralogues are located in one chromosome arm, Chr 13p. In the mouse, however, the three Caspr5 genes are located in two chromosomes, Chr 1 and Chr 17. RT-PCR shows that all three mouse paralogues are being expressed. Common expression is found in brain tissue but different expression patterns are seen in other organs during fetal development and in the adult stage. Tissue specificity of expression has diverged during evolution of this young rodent gene family.     

A mutation in the secretion pathway of the yeast Yarrowia lipolytica that displays synthetic lethality in combination with a mutation affecting the signal recognition particle.

In an attempt to identify proteins involved in the translocation step of protein secretion, a genetic screen was carried out in the yeast Yarrowia lipolytica. A conditional lethal mutant which has a defect in the 7S RNA of the signal recognition particle was mutagenized and screened for second-site mutations that specifically exacerbate its temperature sensitivity. This approach had previously allowed the characterization of an endoplasmic reticulum component, Sls1p, involved in protein translocation. A second mutation, sls2-1, was isolated that causes synthetic lethality when combined with the 7S RNA mutation. On its own, the sls2-1 mutation confers a temperature-sensitive growth phenotype. The secretory phenotype of the sls2 mutant consists in abnormal secretion of several polypeptides, and thus differs from the defect in secretory protein synthesis associated with the 7S RNA and sls1-1 mutations. Two new Y. lipolytica genes were identified which can relieve the growth defect of sls2-1 cells: SLS2 itself and SSL2, a multicopy suppressor of the temperature sensitivity of the sls2 mutant. The SLS2 gene encodes a polypeptide that can potentially be farnesylated and phosphorylated, and shares some homology with an S. cerevisiae protein of unknown function. Ssl2p resembles calmodulin-dependent serine/threonine protein kinases. These two proteins may interact to regulate protein sorting. Received: 9 June 1998 / Accepted: 10 February 1999     

A PMR2 tandem repeat with a modified C-terminus is located downstream from the KRS1 gene encoding lysyl-tRNA synthetase in Saccharomyces cerevisiae

Summary TheKRS1 gene encodes the cytoplasmic form ofSaccharomyces cerevisiae lysyl-tRNA synthetase. TheKRS1 locus has been characterized. The lysyl-tRNA synthetase gene is unique in the yeast genome. The gene is located on the right arm of chromosome IV and disruption of the open reading frame leads to lethality. These results contrast with the situation encountered inEscherichia coli where lysyl-tRNA synthetase is coded by two distinct genes,lysS andlysU, and further address the possible biological significance of this gene duplication. The nucleotide sequence of the 3′-flanking region has been established. It encodes a long open reading frame whose nucleotide and amino acid structures are almost identical toPMR2, a cluster of tandemly repeated genes coding for P-type ion pumps. The sequence alterations relative toPMR2 are mainly located at the C-terminus of the protein.     

Alleles and haplotypes of the MHC-encoded ABC transporters TAP1 and TAP2

TAP1 and TAP2 are two major histocompatibility complex (MHC) genes, located between HLA-DP and -DQ, whose products form a transporter molecule involved in endogenous antigen processing. Polymorphic residues have been described in both genes and, in this study, we have identified another polymorphic site within the adenosine triphosphate (ATP)-binding domain of TAP2. We have used the amplification refractoru mutation system (ARMS) polymerase chain reaction (PCR) to characterize TAP1 and TAP2 alleles and haplotypes in a reference panel of 115 homozygous typing cell lines. Of four possible TAP1 alleles, we observed three, and of eight possible TAP2 alleles, we observed five. Among 88 (homozygous typing cells) (HTCs) homozygous at HLA-DR, -DQ and TP, 80 were also homozygous at TAP1 and TAP2. Of 27 HTCs homozygous at HLA-DR and -DQ, but heterozygous at -DP, 14 were homozygous at TAP1 or TAP2 and 13 heterozygous, consistent with recombination taking place either side of the TAP loci. Of the fifteen possible combinations of TAP1 and TAP2 alleles, we observed eleven, each at a frequency similar to that predicted by individual allele frequencies. In this ethnically heterogeneous panel there is no indication that particular combinations of TAP1 and TAP2 have been maintained together. Correspondence to: S. H. Powis.     

DCPIP (2,6-dichlorophenolindophenol) as a genotype-directed redox chemotherapeutic targeting NQO1*2 breast carcinoma

Abstract

Accumulative experimental evidence suggests feasibility of chemotherapeutic intervention targeting human cancer cells by pharmacological modulation of cellular oxidative stress. Current efforts aim at personalization of redox chemotherapy through identification of predictive tumour genotypes and redox biomarkers. Based on earlier research demonstrating that anti-melanoma activity of the pro-oxidant 2,6-dichlorophenolindophenol (DCPIP) is antagonized by cellular NAD(P)H:quinone oxidoreductase (NQO1) expression, this study tested DCPIP as a genotype-directed redox chemotherapeutic targeting homozygous NQO1*2 breast carcinoma, a common missense genotype [rs1800566 polymorphism; NP_000894.1:p.Pro187Ser] encoding a functionally impaired NQO1 protein. In a panel of cultured breast carcinoma cell lines and NQO1-transfectants with differential NQO1 expression levels, homozygous NQO1*2 MDA-MB231 cells were hypersensitive to DCPIP-induced caspase-independent cell death that occurred after early onset of oxidative stress with glutathione depletion and loss of genomic integrity. Array analysis revealed upregulated expression of oxidative (GSTM3, HMOX1, EGR1), heat shock (HSPA6, HSPA1A, CRYAB) and genotoxic stress response (GADD45A, CDKN1A) genes confirmed by immunoblot detection of HO-1, Hsp70, Hsp70B’, p21 and phospho-p53 (Ser15). In a murine xenograft model of human homozygous NQO1*2-breast carcinoma, systemic administration of DCPIP displayed significant anti-tumour activity, suggesting feasibility of redox chemotherapeutic intervention targeting the NQO1*2 genotype.     

Multiple gene integration to promote erythritol production on glycerol in Yarrowia lipolytica

Objective

Erythritol (1,2,3,4-butanetetrol) is a 4-carbon sugar alcohol that occurs in nature as a metabolite or storage compound. In this study, a multiple gene integration strategy was employed to enhance erythritol production in Y. lipolytica.

Results

The effects on the production of erythritol in Y. lipolytica of seven key genes involved in the erythritol synthesis pathway were evaluated individually, among which transketolase (TKL1) and transaldolase (TAL1) showed important roles in enhancing erythritol production. The combined overexpression of four genes (GUT1, TPI1, TKL1, TAL1) and disruption of the EYD1 gene (encoding erythritol dehydrogenase), resulted in produce approximately 40 g/L erythritol production from glycerol. Further enhanced erythritol synthesis was obtained by overexpressing the RKI1 gene (encoding ribose 5-phosphate isomerase) and the AMPD gene (encoding AMP deaminase), indicating for the first time that these two genes are also related to the enhancement of erythritol production in Y. lipolytica.

Conclusions

A combined gene overexpression strategy was developed to efficiently improve the production of erythritol in Y. lipolytica, suggesting a great capacity and promising potential of this non-conventional yeast in converting glycerol into erythritol.