The Mouse Mitotic Checkpoint Gene Bub1b,a Novel Bub1 Family Member,Is Expressed in a Cell Cycle-Dependent Manner

A search for genes differentially expressed in normal and leukemic mouse thymocytes yielded a homolog of the yeast mitotic checkpoint protein Bub1. This novel protein (“mBub1b”) has 40% sequence similarity to the mouse Bub1 (“mBub1a”) previously described by Taylor and McKeon (1997,Cell 89,727–735) over four extended domains. Differences between the Bub1 sequences suggest that the two proteins may have different substrate specificities and that Bub1b alone has a putative “destruction” box that can target proteins for degradation by proteosomes during mitosis. Northern blots of normal tissues show that mouse Bub1a and Bub1b genes are expressed in thymus and spleen, but not in nondividing tissues. In synchronized cells, expression of both Bub1 genes is undetectable in G₁; Bub1 gene expression peaks in G₂/M with Bub1b delayed by 6 h relative to Bub1a. This cell cycle-dependent expression explains the tissue distribution and the abundance of Bub1 mRNAs in rapidly dividing cell lines. The human equivalent of mBub1b was isolated and mapped to chromosome 15q15. The existence in mammals of two separate Bub1 genes encoding distinct proteins, coupled with the different timing of peak expression, suggests that Bub1a and Bub1b have distinct roles in the mitotic checkpoint.     

The requirement for both DNA polymerase and 5'' to 3'' exonuclease activities of DNA polymerase I during Tn5 transposition

Summary By assaying transposition of Tn5 from b221 cI857 rex::Tn5 (Berg 1977) in PolA-proficient and deficient cells, both the polymerase activity and 5 to 3 exonuclease acivity of DNA polymerase I have been shown to be required for transposition. This requirement could not be observed in three other systems in which the transposon donor replicon had existed in the PolA-proficient and deficient cells before the transposition event to be assayed occurred. By analogy to Tn3, this may indicate that the repressor encoded by Tn5 has already been expressed and hence become rate-limiting in the overall transposition process, even in PolA-deficient cells still possessing a residual activity. One polA mutant was found among more than 50 transposition-deficient (tnp) mutants isolated by the use of b221 cI857 rex::Tn5.     

The human TREX2 3' -> 5'-exonuclease structure suggests a mechanism for efficient nonprocessive DNA catalysis

The 3' --> 5'-exonucleases process DNA ends in many DNA repair pathways of human cells. Determination of the human TREX2 structure is the first of a dimeric 3'-deoxyribonuclease and indicates how this highly efficient nonprocessive enzyme removes nucleotides at DNA 3' termini. Symmetry in the TREX2 dimer positions the active sites at opposite outer edges providing open access for the DNA. Adjacent to each active site is a flexible region containing three arginines positioned appropriately to bind DNA and to control its entry into the active site. Mutation of these three arginines to alanines reduces the DNA binding capacity by approximately 100-fold with no effect on catalysis. The human TREX2 catalytic residues overlay with the bacterial DnaQ family of 3'-exonucleases confirming the structural conservation of the catalytic sites despite limited sequence identity, and mutations of these residues decrease the still measurable activity by approximately 10(5)-fold, confirming their catalytic role.     

The 5'' to 3'' exonuclease activity of DNA polymerase I is essential for Streptococcus pneumoniae

Three different mutations were introduced in the polA gene of Streptococcus pneumoniae by chromosomal transformation. One mutant gene encodes a truncated protein that possesses 5' to 3' exonuclease but has lost polymerase activity. This mutation does not affect cell viability. Other mutated forms of polA that encode proteins with only polymerase activity or with no enzymatic activity could not substitute for the wild-type polA gene in the chromosome unless the 5' to 3' exonuclease domain was encoded elsewhere in the chromosome. Thus, it appears that the 5' to 3' exonuclease activity of the DNA polymerase I is essential for cell viability in S. pneumoniae. Absence of the polymerase domain of DNA polymerase I slightly diminished the ability of S. pneumoniae to repair DNA lesions after ultraviolet irradiation. However, the polymerase domain of the pneumococcal DNA polymerase I gave almost complete complementation of the polA5 mutation in Escherichia coli with respect to resistance to ultraviolet irradiation.     

The Saccharomyces cerevisiae YCC5 (YCL025c) Gene Encodes an Amino Acid Permease, Agp1, Which Transports Asparagine and Glutamine

The yeast YCC5 gene encodes a putative amino acid permease and is homologous to GNP1 (encoding a high-affinity glutamine permease). Using strains with disruptions in the genes for multiple permeases, we demonstrated that Ycc5 (which we have renamed Agp1) is involved in the transport of asparagine and glutamine, performed a kinetic analysis of this activity, and showed that AGP1 expression is subject to nitrogen repression.     

The Drosophila Meiotic Recombination Gene Mei-9 Encodes a Homologue of the Yeast Excision Repair Protein Rad1

Meiotic recombination and DNA repair are mediated by overlapping sets of genes. In the yeast Saccharomyces cerevisiae, many genes required to repair DNA double-strand breaks are also required for meiotic recombination. In contrast, mutations in genes required for nucleotide excision repair (NER) have no detectable effects on meiotic recombination in S. cerevisiae. The Drosophila melanogaster mei-9 gene is unique among known recombination genes in that it is required for both meiotic recombination and NER. We have analyzed the mei-9 gene at the molecular level and found that it encodes a homologue of the S. cerevisiae excision repair protein Rad1, the probable homologue of mammalian XPF/ERCC4. Hence, the predominant process of meiotic recombination in Drosophila proceeds through a pathway that is at least partially distinct from that of S. cerevisiae, in that it requires an NER protein. The biochemical properties of the Rad1 protein allow us to explain the observation that mei-9 mutants suppress reciprocal exchange without suppressing the frequency of gene conversion.     

The Role of Exo1p Exonuclease in DNA End Resection to Generate Gene Conversion Tracts in Saccharomyces cerevisiae

The yeast Exo1p nuclease functions in multiple cellular roles: resection of DNA ends generated during recombination, telomere stability, DNA mismatch repair, and expansion of gaps formed during the repair of UV-induced DNA damage. In this study, we performed high-resolution mapping of spontaneous and UV-induced recombination events between homologs in exo1 strains, comparing the results with spontaneous and UV-induced recombination events in wild-type strains. One important comparison was the lengths of gene conversion tracts. Gene conversion events are usually interpreted as reflecting heteroduplex formation between interacting DNA molecules, followed by repair of mismatches within the heteroduplex. In most models of recombination, the length of the gene conversion tract is a function of the length of single-stranded DNA generated by end resection. Since the Exo1p has an important role in end resection, a reduction in the lengths of gene conversion tracts in exo1 strains was expected. In accordance with this expectation, gene conversion tract lengths associated with spontaneous crossovers in exo1 strains were reduced about twofold relative to wild type. For UV-induced events, conversion tract lengths associated with crossovers were also shorter for the exo1 strain than for the wild-type strain (3.2 and 7.6 kb, respectively). Unexpectedly, however, the lengths of conversion tracts that were unassociated with crossovers were longer in the exo1 strain than in the wild-type strain (6.2 and 4.8 kb, respectively). Alternative models of recombination in which the lengths of conversion tracts are determined by break-induced replication or oversynthesis during strand invasion are proposed to account for these observations.     

1H NMR of 5''CGCGTATATACGCG3'', a duplex and a four-membered loop.

The deoxyribonucleotide 5'CGCGTATATACGCG3' was synthesized and studied by NMR methods. This short, fully palindromic duplex also forms a hairpin under certain conditions described within. As such, it is considered to be a model for cruciform formation. We show that this sequence forms a four-membered loop and a duplex in solution. The duplex is shown to be a normal, B-DNA like helix, while the hairpin is shown to be a four-membered ATAT loop.     

Synthesis and characterization of a ribavirin-3'',5''-phosphate pentadecamer homoribopolymer bearing a 5''-amino tether group and a 3''-thymidine.

The synthesis of a pentadecamer of the 5'-phosphate of the antiviral nucleoside ribavirin (1'-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide) has been achieved. This homoribopolymer is terminated at the 5'-position with an (6-aminohexyl)phosphate group to permit conjugation to a carrier and at the 3'-position by a thymidine-5'-phosphate. The synthesis was accomplished using the methyl phosphoramidite approach to oligoribonucleotides. The homoribopolymer was insensitive to ribonuclease A but was sensitive to ribonuclease T2 digestion.     

The synthesis of protected 5''-amino-2'',5''-dideoxyribonucleoside-3''-O-phosphoramidites; applications of 5''-amino-oligodeoxyribonucleotides.

Synthetic routes to the four appropriately protected 5'-amino-2',5'-dideoxyribonucleoside-3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) have been developed. The structures of all intermediates were confirmed by 13C n.m.r. spectroscopy. These building blocks have been used to prepare 5'-amino-oligodeoxyribonucleotides, which can be coupled to a wide variety of compounds, in particular metal cluster derivatives, but also fluorophores and biotin derivatives, thus generating a variety of very useful probes. Brief mention is made of a tetrairidium cluster derivative of 5'-amino-d[CCGATATCGG], which has been cocrystallised with EcoRV, and will be used for electron microscopy studies.     

The synthesis of protected 5''-mercapto-2'',5''-dideoxyribonucleoside-3''-O-phosphoramidites; uses of 5''-mercapto-oligodeoxyribonucleotides.

The syntheses of the four novel, base protected 5'-(S-triphenylmethyl)mercapto-2',5'-dideoxyribonucleoside-3 '-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) are described. These compounds have been used to prepare 5'-(S-triphenylmethyl) mercapto-oligodeoxyribonucleotides, which are readily purified by reversed phase h.p.l.c., owing to the highly lipophilic trityl group. After cleavage of the S-trityl group by silver or mercuric ions, the free thiol moiety can be coupled to a wide variety of reagents, generating very useful probes. Fluorescent labelled 5'-mercapto-oligodeoxyribonucleotides are being used for automated DNA sequencing without radioactivity, and heavy metal labelled 5'-mercapto-oligonucleotides will be used in X-ray crystallography.     

The Embryonically Active Gene, Unkempt, of Drosophila Encodes a Cys(3)his Finger Protein

The unkempt gene of Drosophila encodes a set of embryonic RNAs, which are abundant during early stages of embryogenesis and are present ubiquitously in most somatic tissues from the syncytial embryo through stage 15 of embryogenesis. Expression of unkempt RNAs becomes restricted predominantly to the central nervous system in stages 16 and early 17. Analysis of cDNAs from this locus reveals the presence of five Cys3His fingers in the protein product. Isolation and analysis of mutations affecting the unkempt gene, including complete deletions of this gene, indicate that there is no zygotic requirement for unkempt during embryogenesis, presumably due to the contribution of maternally supplied RNA, although the gene is essential during post-embryonic development.     

The Caenorhabditis Elegans Sel-1 Gene, a Negative Regulator of Lin-12 and Glp-1, Encodes a Predicted Extracellular Protein

The Caenorhabditis elegans lin-12 and glp-1 genes encode members of the LIN-12/NOTCH family of receptors. The sel-1 gene was identified as an extragenic suppressor of a lin-12 hypomorphic mutant. We show in this report that the sel-1 null phenotype is wild type, except for an apparent elevation in lin-12 and glp-1 activity in sensitized genetic backgrounds, and that this genetic interaction seems to be lin-12 and glp-1 specific. We also find that sel-1 encodes a predicted extracellular protein, with a domain sharing sequence similarity to predicted proteins from humans and yeast. SEL-1 may interact with the LIN-12 and GLP-1 receptors and/or their respective ligands to down-regulate signaling.     

The pbrB Gene Encodes a Laccase Required for DHN-Melanin Synthesis in Conidia of Talaromyces (Penicillium) marneffei

Talaromyces marneffei (Basionym: Penicillium marneffei) is a significant opportunistic fungal pathogen in patients infected with human immunodeficiency virus in Southeast Asia. T. marneffei cells have been shown to become melanized in vivo. Melanins are pigment biopolymers which act as a non-specific protectant against various stressors and which play an important role during virulence in fungi. The synthesis of the two most commonly found melanins in fungi, the eumelanin DOPA-melanin and the allomelanin DHN-melanin, requires the action of laccase enzymes. The T. marneffei genome encodes a number of laccases and this study describes the characterization of one of these, pbrB, during growth and development. A strain carrying a PbrB-GFP fusion shows that pbrB is expressed at high levels during asexual development (conidiation) but not in cells growing vegetatively. The pbrB gene is required for the synthesis of DHN-melanin in conidia and when deleted results in brown pigmented conidia, in contrast to the green conidia of the wild type.     

The 3'' to 5'' exonuclease activity located in the DNA polymerase delta subunit of Saccharomyces cerevisiae is required for accurate replication.

In Saccharomyces cerevisiae, DNA polymerase delta (POLIII), the product of the CDC2 (POL3) gene, possesses, in its N-terminal half, the well conserved 3-domain 3' to 5' exonuclease site. Strains selectively mutagenized in this site display a mutator phenotype detected as a drastically increased spontaneous forward mutation rate to canavanine resistance or as an elevated reversion rate to lysine prototrophy. Assays on a partially purified extract of the mutant giving the largest mutator effect indicate that the 3' to 5' exonuclease activity is reduced below the detection limit whereas the DNA polymerizing activity has wild-type level. Therefore, our results provide experimental support for the hypothesis that the exonucleolytic proofreading activity associated with DNA polymerase delta resides on the DNA polymerase delta subunit and enhances the fidelity of DNA replication in yeast.     

The Saccharomyces cerevisiae MLF6/YPL244C Gene, Which Encodes a Possible Yeast Homologue of Mammalian UDP-Galactose Transporter, Confers Resistance to the Immunosuppressive Drug, Leflunomide

The immunosuppressant leflunomide (LFM) inhibits the growth of cytokine-stimulated proliferation of lymphoid cells in vitro and also inhibits the growth of eukaryotic microorganism, Saccharomyces cerevisiae. As a first step to elucidate the molecular mechanism of LFM action in human, yeast gene which suppresses the anti-proliferative effect when in increased copy number was cloned and designated MLF6 for multicopy suppressor of LFM sensitivity. DNA sequencing analysis revealed that the MLF6 gene is identical to the YPL244C gene which encodes a possible yeast homologue of human UDP-galactose transporter. The disruption of the MLF6 gene increased the sensitivity of yeast cells to the drug. Received: 13 October 2000 / Accepted: 4 January 2001     

The Arabidopsis LOS5/ABA3 Locus Encodes a Molybdenum Cofactor Sulfurase and Modulates Cold Stress– and Osmotic Stress–Responsive Gene Expression

To understand low temperature and osmotic stress signaling in plants, we isolated and characterized two allelic Arabidopsis mutants, los5-1 and los5-2, which are impaired in gene induction by cold and osmotic stresses. Expression of RD29A-LUC (the firefly luciferase reporter gene under the control of the stress-responsive RD29A promoter) in response to cold and salt/drought is reduced in the los5 mutants, but the response to abscisic acid (ABA) remains unaltered. RNA gel blot analysis indicates that the los5 mutation reduces the induction of several stress-responsive genes by cold and severely diminishes or even completely blocks the induction of RD29A, COR15, COR47, RD22, and P5CS by osmotic stresses. los5 mutant plants are compromised in their tolerance to freezing, salt, or drought stress. los5 plants are ABA deficient, as indicated by increased transpirational water loss and reduced accumulation of ABA under drought stress in the mutant. A comparison with another ABA-deficient mutant, aba1, reveals that the impaired low-temperature gene regulation is specific to the los5 mutation. Genetic tests suggest that los5 is allelic to aba3. Map-based cloning reveals that LOS5/ABA3 encodes a molybdenum cofactor (MoCo) sulfurase. MoCo sulfurase catalyzes the generation of the sulfurylated form of MoCo, a cofactor required by aldehyde oxidase that functions in the last step of ABA biosynthesis in plants. The LOS5/ABA3 gene is expressed ubiquitously in different plant parts, and the expression level increases in response to drought, salt, or ABA treatment. Our results show that LOS5/ABA3 is a key regulator of ABA biosynthesis, stress-responsive gene expression, and stress tolerance.