Choice of an adequate promoter for efficient complementation in Saccharomyces cerevisiae: a case study

Conservation of the function of open reading frames recently identified in fungal genome projects can be assessed by complementation of deletion mutants of putative Saccharomyces cerevisiae orthologs. A parallel complementation assay expressing the homologous wild type S. cerevisiae gene is generally performed as a positive control. However, we and others have found that failure of complementation can occur in this case. We investigated the specific cases of S. cerevisiae TBF1 and TIM54 essential genes. Heterologous complementation with Candida glabrata TBF1 or TIM54 gene was successful using the constitutive promoters TDH3 and TEF. In contrast, homologous complementation with S. cerevisiae TBF1 or TIM54 genes failed using these promoters, and was successful only using the natural promoters of these genes. The reduced growth rate of S. cerevisiae complemented with C. glabrata TBF1 or TIM54 suggested a diminished functionality of the heterologous proteins compared to the homologous proteins. The requirement of the homologous gene for the natural promoter was alleviated for TBF1 when complementation was assayed in the absence of sporulation and germination, and for TIM54 when two regions of the protein presumably responsible for a unique translocation pathway of the TIM54 protein into the mitochondrial membrane were deleted. Our results demonstrate that the use of different promoters may prove necessary to obtain successful complementation, with use of the natural promoter being the best approach for homologous complementation.     

The chloroplast gene cluster containing psbF,psbL, petG and rps3 is conserved in Chlamydomonas

We have sequenced a 6.8-kb segment of the Chlamydomonas eugametos chloroplast DNA which contains the psbF, psbL, petG and rps3 genes. As in the distantly related green alga Chlamydomonas reinhardtii, these genes reside in this order (53) on the same DNA strand, suggesting that such a chloroplast gene cluster was present in the most recent common ancestor of all Chlamydomonas species. For each of the four genes, with the exception of rps3, the C. eugametos and C. reinhardtii coding regions were found to be identical, or very similar, in length, whereas each of the intergenic spacers is substantially longer in C. eugametos than in C. reinhardtii. The central portion of both Chlamydomonas rps3 genes features a long extra coding region relative to other rps3 sequences. We have shown that the insertion sequence in the C. eugametos rps3 is not excised at the RNA level.     

Genetic evidence for different RAD52-dependent intrachromosomal recombination pathways in Saccharomyces cerevisiae

Mutations in the RecA-like genes RAD51 and RAD57 reduce the frequency of gene conversion/reciprocal exchange between inverted repeats 7-fold. However, they enhance the frequency of deletions between direct repeats 5–12-fold. These induced deletions are RAD1- and RAD52-dependent. On the basis of these results it is proposed that there are several RAD52-dependent pathways of recombination: the recombinational repair pathway of gene conversion/reciprocal exchange dependent on RAD51 and RAD57; a RAD1-and RAD52-dependent pathway exclusively responsible for deletions that are induced in rad51 and rad57 mutants; and finally, it is possible that the gene conversion/reciprocal exchange events observed in rad51 and rad57 strains represent another RAD52-dependent recombination pathway of gene conversion/reciprocal exchange that does not require Rad51 and Rad57 functions. It is also shown that the RAD10 excision-repair gene is involved in long gene conversion tracts in homologous recombination between inverted repeats, as previously observed for RAD1. Finally, an analysis of meiotic recombination reveals that deletions are induced in meiosis 100-fold above mitotic levels, similar to intrachromosomal gene conversion/reciprocal exchange, and that, in contrast to intrachromosomal meiotic gene conversion (50% association), intrachromosomal meiotic gene conversion is not preferentially associated with reciprocal exchange (12–30% of association).     

Mutations in the structural gene for cytochrome c result in deficiency of both cytochromes aa 3 and c in Neurospora crassa

The cyt-12-12 mutant of Neurospora crassa is characterized by slow growth and a deficiency of spectrophotometrically-detectable cytochromes aa ₃ and c. Using a sib-selection procedure we have isolated the cyt-12 ⁺ allele from a cosmid library of N. crassa genomic DNA. Characterization of the cyt-12 ⁺ allele reveals that it encodes the structural gene for cytochrome c. DNA sequence analysis of the cyt-12-12 allele revealed a mutation in the cytochrome c coding sequence that results in replacement of a glycine residue, which is invariant in the cytochrome c of other species, with an aspartic acid. Genetic analysis confirms that cyt-12-12 is allelic with the previously-characterized cyc-1-1 mutant, which was also shown to affect the single locus encoding cytochrome c in N. crassa. We suggest that the amount of functional cytochrome c present in mitochondria influences the level of cytochrome aa ₃ .     

Promoter Methylations of p16 and p14 Genes in Early and Advanced Gastric Cancer. Correlations of the Modes of Their Occurrence with Histologic Type

The INK4a/ARF locus encodes two cell cycle-regulatory proteins, p16^INK4a and p14^ARF. These share an exon using different reading frames, and act through Rb and p53 pathways. Recently, it has been found that silencing of p16^INK4a and p14^ARF expressions by aberrant methylation of the CpG islands in the promoter regions is an alternative mechanism that inactivates possible tumor suppressor functions in various tumors. To clarify the features of gastric cancers with promoter methylation of p16^INK4a and p14^ARF, we investigated the methylation status in gastric cancer cell lines and primary gastric cancers using methylation-specific PCR (MSP), and correlated the methylation status with microsatellite instability (MSI), DNA ploidy pattern, p53 immunohistochemistry, and various clinicopathologic factors, paying attention to the correlations with the histologic types. Of 10 cell lines studied, silencing of the expression of p16^INK4a and p14^ARF due to promoter methylation was detected by MSP and RT-PCR in six (60%) and two (20%) cell lines, respectively. p14^ARF silencing was detected only in cell lines derived from gastric cancer of the diffuse type, while p16^INK4a silencing was found in cell lines derived from both diffuse and intestinal types. In 59 primary gastric cancers, promoter methylation of p16^INK4a and p14^ARF was found in 10 (17%) and 14 (24%) of the tumors independently, there being an association with DNA diploidy, but not with p53 immunohistochemistry. p16^INK4a methylation was found irrespective of tumor stages and histology. Whereas p14^ARF methylation was found more frequently in intestinal type cancers in an early stage and in diffuse type cancers in an advanced stage, MSI tended to be related especially to p14^ARF methylation in cancers of the intestinal type. Thus, the significance of p14^ARF methylation differed between intestinal and diffuse types, while such a difference was not observed in p16^INK4a methylation.     

Increased expression of the CD80 accessory molecule by alveolar macrophages in asthmatic subjects and its functional involvement in allergen presentation to autologous TH2 lymphocytes

Background: Alveolar macrophages (AMs) are more efficient antigen-presenting cells in allergic individuals than in nonatopic subjects. Objective: We studied whether this difference may be correlated to increased expression of membrane costimulatory molecules, such as the B7 molecules (CD80 and CD86). Methods: Eleven subjects with allergic asthma sensitized to Dermatophagoides pteronyssinus and 5 healthy nonatopic volunteers underwent bronchoalveolar lavage, and the costimulatory molecule expression on AMs was evaluated. Peripheral blood T cells, either freshly isolated or as established D pteronyssinus -specific cell lines, were cultured with autologous monocytes or AMs as antigen-presenting cells. In vitro allergen-induced proliferation and cytokine production were evaluated in the presence of B7-blocking reagents. Results: Allergic individuals had a significantly higher proportion of AMs expressing the CD80 molecule than control subjects (28.5% ± 14.8% vs 1.4% ± 1.2%; P < .001), whereas no difference was observed in CD86 expression (2.0% ± 2.3% vs 1.1% ± 0.6; P > .1). In a large proportion of the asthmatic subjects we studied, AMs were presenting soluble antigens (tetanus toxoid and streptolysin-O) to freshly isolated T cells more efficiently than AMs from nonatopic control subjects. Finally, both T-cell proliferation and cytokine production of D pteronyssinus- specific established T-cell lines were inhibited by a CD80-blocking antibody in a dose-dependent manner. Conclusion: Costimulation by means of CD80 expressed by AMs is probably involved in the amplification of the allergen-specific T-lymphocyte response in the airways of asthmatic subjects. (J Allergy Clin Immunol 1999;103:1136-42.)     

Identification and characterization of odv-e25 of Spodoptera litura multicapsid nucleopolyhedrovirus

Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) odv-e25 is 684 bp long, potentially encoding 227 amino acids with a predicted molecular weight of 24.9 kDa. Homology analysis indicated that SpltMNPV ODV-E25 has 35–65% amino acid identity with that of other known baculoviruses. RT-PCR results revealed that the odv-e25 is transcribed actively at the late stage of infection and the mRNA start site was mapped within a consensus baculovirus late promoter sequence (TTAAG). Western blot analysis of odv-e25 expression with an antiserum made against 6 × His tagged ODV-E25 expressed in Escherichia coli indicated that it was present as a doublet of approximately 27 kDa from 24 h through 96 h in SpltMNPV-infected Spli-221 cells. Similar results were seen on Western blots of Spodoptera exigua (Se)MNPV-infected Se301 cells. Immunofluorescence analysis showed that ODV-E25 was predominantly present in the cytoplasm of SpltMNPV-infected cells and localized to the envelopes of occlusion-derived virus.     

Cooperative effect of two amino acid mutations in the coat protein of Pepper mild mottle virus overcomes L3-mediated resistance in Capsicum plants

We found that an L ³ resistance-breaking field isolate of Pepper mild mottle virus (PMMoV), designated PMMoV-Is, had two amino acid changes in its coat protein (CP), namely leucine to phenylalanine at position 13 (L13F) and glycine to valine at position 66 (G66V), as compared with PMMoV-J, which induces a resistance response in L ³ -harboring Capsicum plants. The mutations were located to a CP domain corresponding to the outer surface of PMMoV particles in computational molecular modeling. Analyses of PMMoV CP mutants containing either or both of these amino acid changes revealed that both changes were required to efficiently overcome L ³ -mediated resistance with systemic necrosis induction. Although CP mutants containing either L13F or G66V could not efficiently overcome L ³ -mediated resistance, these amino acid changes had different effects on the elicitor activity of PMMoV CP. L13F caused a slight reduction in the elicitor activity, resulting in virus restriction to necrotic local lesions that were apparently larger than those induced by wild-type PMMoV, while G66V rendered wild-type PMMoV the ability to overcome L ³ -mediated resistance, albeit with a lower efficiency than PMMoV with both changes. These results suggest that a cooperative effect of the L13F and G66V mutations on the elicitor activity of CP is responsible for overcoming the L ³ -mediated resistance.     

A new enrichment approach identifies genes that alter cell cycle progression in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Mechanisms that coordinate cell growth with division are thought to determine the timing of initiation of cell division and to limit overall cell proliferation. To identify genes involved in this process in Saccharomyces cerevisiae, we describe a method that does not rely on cell size alterations or resistance to pheromone. Instead, our approach was based on the cell surface deposition of the Flo1p protein in cells having passed START. We found that over-expression of HXT11 (which encodes a plasma membrane transporter), PPE1 (coding for a protein methyl esterase), or SIK1 (which encodes a protein involved in rRNA processing) shortened the duration of the G1 phase of the cell cycle, prior to the initiation of DNA replication. In addition, we found that, although SIK1 was not part of a mitotic checkpoint, SIK1 over-expression caused spindle orientation defects and sensitized G2/M checkpoint mutant cells. Thus, unlike HXT11 and PPE1, SIK1 over-expression is also associated with mitotic functions. Overall, we used a novel enrichment approach and identified genes that were not previously associated with cell cycle progression. This approach can be extended to other organisms.     

Clinical and molecular characterization of 40 patients with Noonan syndrome

Noonan syndrome (NS, OMIM 163950) is an autosomal dominant disorder, with a prevalence at birth of 1:1000–1:2500 live births, characterized by short stature, facial and skeletal dysmorphisms, cardiovascular defects and haematological anomalies. Missense mutations of PTPN11 gene account for approximately 50% of NS cases, while molecular lesions of other genes of the RAS/MAPK pathway – KRAS, SOS1 and RAF1 – play a minor role in the molecular pathogenesis of the disease. Forty patients were enrolled in the study with a PTPN11 mutation detection rate of 31.5%, including a novel missense mutation, Phe285Ile, in a familial case with high intrafamilial phenotypic variability. All patients negative for PTPN11 mutations were further screened for mutations of the KRAS, SOS1, and RAF1 genes, revealing a Thr266Lys substitution in SOS1 in a single patient, a newborn with a subtle phenotype, characterized by facial dysmorphisms and a mild pulmonic stenosis.     

DFNB66 and DFNB67 loci are non allelic and rarely contribute to autosomal recessive nonsyndromic hearing loss

We previously mapped the DFNB66 locus to an interval overlapping the DFNB67 region. Mutations in the LHFPL5 gene were identified as a cause of DFNB67 hearing loss (HL). However, screening of the coding exons of LHFPL5 did not reveal any mutation in the DFNB66 family. The objective of this study was to check whether DFNB66 and DFNB67 are distinctive loci and determining their contribution to HL. In the DFNB66 family, sequencing showed absence of mutations in the untranslated regions and the predicted promoter sequence of LHFPL5. Analysis of five microsatellites in the 6p21.31–22.3 region and screening of the LHFPL5 gene by DNA heteroduplex analysis in DHPLC revealed a novel mutation (c.89dup) in one out of 129 unrelated Tunisian families with autosomal recessive nonsyndromic (ARNS) HL. Our findings suggest that two distinct genes are responsible for DFNB66 and DFNB67 HL. These loci are likely to be a rare cause of ARNSHL.     

Molecular screening of patients with nonsyndromic hearing loss from Nanjing city of China

Hearing loss is the most frequent sensory disorder involving a multitude of factors,and at least 50% of cases are due to genetic etiology.To further characterize the molecular etiology of hearing loss in the Chinese population,we recruited a total of 135 unrelated patients with nonsyndromic sensorineural hearing loss (NSHL) for mutational screening of GJB2,GJB3,GJB6,SLC26A4,SLC26A5 IVS2-2A>G and mitochondrial 12SrRNA,tRNA Ser(UCN) by PCR amplification and direct DNA sequencing.The carrier frequencies of deafness-causing mutations in these patients were 35.55% in GJB2,3.70% in GJB6,15.56% in SLC26A4 and 8.14% in mitochondrial 12SrRNA,respectively.The results indicate the necessity of genetic screening for mutations of these causative genes in Chinese population with nonsyndromic hearing loss.     

Effect of AA-2414, a thromboxane A2 receptor antagonist, on airway inflammation in subjects with asthma

Background: Asthma is a chronic inflammatory disease of the airways. The chemokines are potent chemoattractants for eosinophils and other types of cells associated with allergic inflammation. AA-2414, a new thromboxane A₂ receptor antagonist, reduces bronchial hyperresponsiveness in asthmatic subjects, but its mechanism of action is unclear. Objective: We tested the hypothesis that the beneficial effects of AA-2414 in asthma result from reduction in the number of inflammatory cells infiltrating the airway associated with inhibition of chemokine release. Methods: We studied bronchial biopsy specimens from 31 asthmatic subjects before and after oral treatment with AA-2414 (80 mg/day) or matched placebo for 4 months in a double-blind manner. Biopsy specimens were examined by immunohistochemistry. Each subject recorded symptom score and peak expiratory flow (PEF). Lung function and bronchial responsiveness to methacholine were measured before and after treatment. Results: After treatment, significant improvements in symptom score (P < .05), PEF (P < .01), diurnal variation of PEF (P < .01), and bronchial responsiveness (P < .01) were observed in the AA-2414 group compared with the placebo group. These improvements were accompanied by a significant decrease in the number of submucosal EG2⁺ eosinophils (P < .05). There was also a reduction in the number of cells expressing RANTES (P < .05) and macrophage inflammatory protein (MIP)-1α (P < .05) in the epithelium and of cells expressing monocyte chemotactic protein-3 (P < .01), RANTES (P < .05), MIP-1α (P < .01), and eotaxin (P < .01) in the submucosa in the AA-2414 treatment group. A significant correlation was found between the number of EG2⁺ eosinophils and numbers of monocyte chemotactic protein-3⁺ (r_s = 0.52, P < .005), MIP-1α⁺ (r_s = 0.34, P < .05), and eotaxin⁺ cells (r_s = 0.47, P < .01) in the submucosa. There was a significant negative correlation between the increase in bronchial responsiveness and the change in number of submucosal EG2⁺ cells (r_s = –0.65, P < .001). Conclusions: These findings suggest that AA-2414 treatment of patients with asthma may inhibit activated eosinophil infiltration in part by modulating the expression of chemokines in bronchial tissues. (J Allergy Clin Immunol 1999;103:1054-61.)     

Association between polymorphisms in the biometabolism genes CYP1A1, GSTM1, GSTT1 and GSTP1 in bladder cancer

Numerous enzymes, including Cytochrome P450s (phase I) and Glutathione-S-transferases (phase II), are involved in the metabolic activation and detoxification of carcinogens. Epidemiological studies have consistently demonstrated that bladder cancer is strongly associated with cigarette smoking, and the risk for the development of this neoplasia may be modified by individual differences in carcinogen-metabolizing genes. We investigated the relationship between polymorphisms in the CYP1A1, GSTM1, GSTT1, and GSTP1 genes in a case–control study with 100 bladder cancer patients and 100 controls matched for age, gender, race, and smoking status. The GSTM1, GSTT1, CYP1A1 (A2455→G), and GSTP1 (A313→G) genotypes were determined using a multiplex PCR, an allele specific PCR, and a restriction fragment length polymorphism-PCR method. The present case–controlled association study did not detect any positive or negative association for the GSTM1 and GSTP1 genes [odds ratios (OR) = 1.35; 95% confidence interval (CI) = 0.76–2.41 and OR = 0.75; 95% CI = 0.41–1.38, respectively]. Notably, the genes GSTT1 and CYP1A1 exhibited a statistically significant association with bladder cancer (OR = 1.77; 95% CI = 1.01–3.12 and OR = 1.99; 95% CI = 1.07–3.73). No differences for GSTM1 and GSTP1 genotype prevalence between the bladder cancer cases and the controls were observed, however, the null genotype for the GSTT1 gene and the A/G and G/G variants of the CYP1A1 gene may contribute to the development of bladder cancer.     

Organization and structure of plastome psbF,psbL, petG and ORF712 genes in Chlamydomonas reinhardtii

Summary We have determined the nucleotide sequence of a 5159 base-pair (bp) region of the Chlamydomonas reinhardtii plastome containing three photoelectron transport genes, psbF, psbL and petG, and an unusual open reading frame, ORF712. The photosynthetic genes have an unprecedented arrangement. psbF and psbL are located in close proximity to petG, and are not grouped with two other genes of the cytochrome b₅₅₉ locus, psbE and ORF42. ORF712, located adjacent to psbL, has homology at its 5-and 3-ends to the ribosomal protein rps3 gene, but contains, a central 437 residue domain that lacks similarity to any other known sequence. These sequences add to the growing body of evidence that the chloroplast genome of C. reinhardtii has a significantly different gene arrangement to its counterpart in plants. The structure of ORF712 also provides another example of a phenomenon we have discovered with C. reinhardtii RNA polymerase genes (Fong and Surzycki 1992); namely, that the algal plastome contains chimeric genes in which reading frames with homology to known genes are juxtaposed in-frame with long coding regions of unknown identity.     

The role of opsonization ofStaphylococcus aureus in the development of local inflammation and system response

Thein vitro neutrophil-stimulating activities of twoS. aureus strains are compared with theirin vivo cytotoxic activities, including the use of intact heterologous neutrophils. After opsonization with normal autologous serum, clinical isolates ofS. aureus differ in the ability to induce luminol-dependent chemiluminescence of guinea pig peritoneal neutrophils. After opsonization, the opsonin-dependent strain markedly stimulates chemiluminescence in comparison with the opsonin-independent strain. The local inflammation induced in guinea pig by intracutaneous administration of the opsonized opsonin-dependent strain is more intense than that induced by the opsonin-independent strain. Intramuscular administration of opsonin-dependentS. aureus strain increases mortality in mice from 10 to 46% while the addition of normal guinea pig neutrophils to the inoculate has no effect on this process. Opsonization of opsonin-independent strain decreased mortality from 78 to 40%, the effect being potentiated by the addition of neutrophils to inoculate (mortality 14%). Presumably, the opsonin dependence ofS. aureus manifestedin vitro is associated with its pathogenicityin vivo, which may be caused by intense stimulation of the respiratory burst in neutrophils. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 9, pp. 298–300, September, 1996     

Propagation of the [<Emphasis Type="Italic">PIN</Emphasis><Superscript><Emphasis Type="Italic">+</Emphasis></Superscript>] prion by fragments of Rnq1 fused to GFP

Prions are viewed as enigmatic infectious entities whose genetic properties are enciphered solely in an array of self-propagating protein aggregate conformations. Rnq1, a yeast protein with yet unknown function, forms a prion named [PIN ⁺ ] for its ability to facilitate the de novo induction of another prion, [PSI ⁺ ]. Here we investigate a set of RNQ1 truncations that were designed to cover major Rnq1 sequence elements similar to those important for the propagation of other yeast prions: a region rich in asparagines and glutamines and several types of oligopeptide repeats. Proteins encoded by these RNQ1 truncations were tested for their ability to (a) join (decorate) pre-existing [PIN ⁺ ] aggregates made of wild-type Rnq1 and (b) maintain the heritable aggregated state in the absence of wild-type RNQ1. While the possible involvement of particular sequence elements in the propagation of [PIN ⁺ ] is discussed, the major result is that the efficiency of transmission of [PIN ⁺ ] from wild-type Rnq1 to a fragment decreased with the fragment’s length. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mobilization and evolutionary history of miniature inverted-repeat transposable elements (MITEs) in Beta vulgaris L.

We have identified three families of miniature inverted-repeat transposable elements (VulMITEs) in the genome of sugar beet (Beta vulgaris L.), evidently derived from a member of the Vulmar family of mariner transposons. While VulMITEs I are typical stowaway-like MITEs, VulMITEs II and VulMITEs III are rearranged stowaway elements of increased size. The integration of divergent moderately and highly repetitive sequences into VulMITEs II and, in particular in VulMITEs III, respectively, shows that amplification of repetitive DNA by MITEs contribute to the increase of genome size with possible implications for plant genome evolution. Fluorescent in-situ hybridization (FISH), for the first time visualizing stowaway MITE distribution on plant chromosomes, revealed a dispersed localization of VulMITEs along all B. vulgaris chromosomes. Analysis of the flanking sequences identified a dispersed repeat as target site for the integration of the stowaway element VulMITE I. Recent transposition of VulMITE I, which most likely occurred during the domestication of cultivated beets, was concluded from insertional polymorphisms between different B. vulgaris cultivars and species. ^†Sequence data from this article have been deposited in the EMBL/GenBank Data Library under the accession nos. AM231630-AM231653 and AM259123-AM259125.     

Cloning of the Rhizopus niveus pyr4 gene and its use for the transformation of Rhizopus delemar

We have cloned a pyr4 gene encoding orotidine-5-monophosphate decarboxylase of the filamentous fungus Rhizopus niveus. The pyr4 gene of R. nivens has an open reading frame composed of 265 amino-acid residues and has two putative introns. We have also isolated a pyr4 mutant of Rhizopus delemar from 5-fluoroorotic acid-resistant mutants and transformed it with the pyr4 gene of R. niveus as a selectable marker. Introduced DNA was integrated into the chromosome in a multiple tandem array. The mitotic stability of the introduced DNA was increased by a repeated sporulation process. The expression of the Escherichia coli -glucuronidase gene in R. delemar was successfully obtained under the control of the pgk2 gene promoter of R. niveus by co-transformation with the pyr4 gene.     

Cloning and characterization of the SKI3 gene of Saccharomyces cerevisiae demonstrates allelism to SKI5

Summary We have identified a mutant strain of the yeast Saccharomyces cerevisiae which overproduces killer toxin. This strain contains a single mutation which fails to complement defects in both the SKI3 and SKI5 genes, while a cloned copy of this gene complements both the ski3 and ski5 defects. The level of secreted toxin from a cDNA based plasmid is not increased in a ski3 strain, showing that the overproduction phenotype is dependent upon an increased level of M1 dsRNA.