The family of mouse phosphoglycerate kinase genes and pseudogenes

The mammalian genome contains two genes encodingphosphoglycerate kinase; the pgk-1gene is X-linked and is expressed in all cells except sperm, while the pgk-2gene is expressed exclusively in sperm cells. The mouse genome contains no pseudogenes derived from pgk-2.On the other hand, the genomes of Balb/c and C3H/He strain mice contain six other regions with sequences homologous to those of pgk-1cDNA. These pgk-related sequences are likely derived from the pgk-1gene by retroposition because all are located on autosomal chromosomes and because none appear to be interrupted by introns. Two of the presumed pseudogenes contain sequences homologous to all regions of the pgk-1cDNA while the other four genomic regions were truncated at the 5, 3, or both ends. One of the truncated pseudogenes was sequenced. Its pgk-related sequence was not flanked by direct repeats, suggesting that loss of the 5 and/or 3 ends of this retrogene may have occurred following its integration into the genome. Our evidence suggests that pgk-1-derived retroposons arose initially more than 100 million years ago and have continued to arise until so recently that some are unique to different mouse strains.     

Genetic characterization of an α-specific gene responsible for sexual agglutinability in Saccharomyces cerevisiae: mapping and gene dose effect

Summary A recessive ag1 mutation leads to specific defect in sexual agglutinability specifically in cells of the yeast Saccharomyces cerevisiae. The cryptopleurine resistance gene cry^R 1, closely linked to the mating type locus, was used to select / strains which emerged from / strains by mitotic nonreciprocal recombination, to genetically analyse ag1, since ag1 is expressed only in mating type. The ag1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv3 at 12 cM on chromosome X. Sexual agglutinability of cells was shown to be dependent on the dose of the AG1 gene, using / isogenic strains carrying AG1/AG1, AG1/ag1 or ag1/ag1. The sst2-1 mutation did not suppress the ag1 mutation. Based on these results, function of the AG1 gene is discussed.Abbreviations cM centimorgan - FDS first division segregation - NPD nonparental ditype - PD parental ditype - SDS second division segregation - TT tetratype     

Β-Adrenoceptor density on mononuclear leukocytes and right atrial myocardium in infants and children with congenital heart disease

Summary Sympathetic regulation of myocardial performance has been shown to be altered in congestive heart failure. Right atrial tissue of children with severe acyanotic and cyanotic congenital heart disease (CHD) showed a significantly lower-receptor density than that of children with less severe defects. Since mononuclear leukocytes (MNL) contain a homogeneous population of ₂-adrenoceptors which have similar properties to those of cardiac ₂-adrenoceptors, they are frequently used for studying the-adrenergic system. In a group of 37 children with CHD of different types and severity who underwent cardiac surgery, we compared the MNL-adrenoceptor density to the type and severity of CHD and looked for a possible relationship to plasma catecholamine levels and to the right atrial-adrenoceptor density. Membranes of MNL and myocardial cells were radiolabeled with (–)3-[¹²⁵I]Iodocyanopindolol ([¹²⁵I]ICYP). A significantly higher-adrenoceptor density on MNL was found in patients with moderate acyanotic CHD (group I) than in those with severe acyanotic (group II) and cyanotic CHD (group III). Patients of group I showed approximatively 50% higher myocardial-receptor density than those of groups II and III. ICI 118.551-[¹²⁵I]ICYP competition studies revealed that in groups II and III significantly lower proportions and densities of ₁-receptors were found compared to group I. Noradrenaline (NA) plasma levels in group II and group III were significantly higher than those in group I. The adrenaline plasma levels were found to be very high in all children with CHD. A significant negative correlation between NA levels and myocardial total and ₁-adrenoceptor density, but no correlation between plasma catecholamines and MNL-adrenoceptor density, was calculated. We conclude that modulation of MNL-adrenoceptors is not simply controlled by circulating catecholamine levels. Cardiac ₂-adrenoceptor density remained unaltered, but the ₁-density was significantly lowered. ₂-adrenoceptors on MNL showed a slight but significant decrease. However, cardiac ₂-adrenoceptor density cannot be predicted by measuring the-adrenoceptor density on MNL.Abbreviations CHD Congenital heart disease - MNL mononuclear leukocytes - NA noradrenaline - P_PA/P_AO Ratio between pulmonary and aortic pressure - Qp/Qs Ratio between pulmonary and systemic blood flow - HPLC-ED High performance liquid chromatography with electrochemical detector     

<Emphasis Type="Italic">Bgl</Emphasis>II gene polymorphism of the α2β1 integrin gene is a risk factor for diabetic retinopathy in Caucasians with type 2 diabetes

Platelets are thought to be involved in the pathogenesis of diabetic retinopathy. The BglII gene polymorphism of the 21 integrin, which is a platelet collagen receptor, has been suggested as a genetic risk factor for diabetic retinopathy in Japanese subjects. The aim of this study was to look for a relationship between the BglII gene polymorphism of the 21 integrin gene and the development of diabetic retinopathy in Caucasians with type 2 diabetes. Subjects with type 2 diabetes and diabetic retinopathy (n=163) were compared with diabetic subjects without diabetic retinopathy (n=95). A significantly higher frequency of the BglII (+/+) genotype of the gene polymorphism of the 21 integrin gene was found in patients with diabetic retinopathy compared with patients without diabetic retinopathy (19.6% vs 7.4%; P=0.008). The present study demonstrates that the BglII (+/+) genotype of the gene polymorphism of the 21 integrin gene is an independent risk factor (odds ratio: 2.4, 95% confidence interval 1.0–6.0; P<0.05) for diabetic retinopathy in Caucasians with type 2 diabetes.     

Using denaturing HPLC for SNP discovery and genotyping,and establishing the linkage disequilibrium pattern for the all-<Emphasis Type="Italic">trans</Emphasis>-retinol dehydrogenase (<Emphasis Type="Italic">RDH8</Emphasis>) gene

All-trans-retinol dehydrogenase (RDH8) is a visual cycle enzyme that reduces all-trans-retinal to all-trans-retinol. As part of an on-going effort to map genes involved in complex eye diseases, myopia in particular, using association studies, single nucleotide polymorphisms (SNPs) were identified and linkage disequilibrium (LD) pattern was established within and around the RDH8 gene. We used denaturing high-performance liquid chromatography (DHPLC) to screen SNPs in four DNA pools each consisting of DNA from five individuals and genotyped the identified SNPs in 150 Chinese subjects from Hong Kong. Fifteen SNPs were identified: seven were common with the minor allele frequency >0.05 and ten were novel. Common SNPs were included in LD and haplotype analysis using the ASSOCIATE and EH programmes. Four SNPs in the 3 region exhibited significant LD and formed a haplotype block, while three common SNPs in the 5 region did not exhibit useful LD. The LD pattern around the RDH8 gene suggested that one SNP from the 3region and two to three SNPs from the 5 region were needed in association studies involving RDH8. Our results demonstrated the efficiency of DHPLC in screening SNPs when coupled with DNA pooling strategy and in genotyping SNPs.     

‘Phase variation’-type regulation of gene expression and gene replacement mediated by FLP recombinase in the yeast Saccharomyces cerevisiae

Expression of a neomycin phosphotransferase II (NPTII) gene has been designed to be regulated by an FLP-mediated switching of the orientation of the NPTII coding region located on the invertible DNA segment in episomal yeast plasmids. Inversion of the segment from inverted to direct orientation with respect to the promoter resulted in a dramatic increase in G418 resistance. FLP also promoted a double reciprocal exchange between the transforming and the resident 2-m plasmid, leading to insertion of the FLP and REP2 genes into the transforming plasmid. The results demonstrate a possible use of FLP recombinase for phase variation-type regulation of gene expression and gene replacement in eukaryotic cells.     

Binding of human fibrinogen and its polypeptide chains to group B streptococci

Of 51 group-B streptococcal cultures, 20 bound¹²⁵I-labelled human fibrinogen. In contrast to the streptococci of groups A, C and G, the group-B streptococci interacted more with the, -chain of fibrinogen than with whole fibrinogen. None of the streptococcal cultures reacted with-chain. The fibrinogen-negative group-B streptococci still bound the, -chain. The binding of¹²⁵I-labelled,, -chain could be inhibited by unlabelled fibrinogen with fibrinogen-positive, but not with fibrinogen-negative streptococci of group B.     

NADH-reductive stress in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> induces the expression of the minor isoform of glyceraldehyde-3-phosphate dehydrogenase (<Emphasis Type="Italic">TDH1</Emphasis>)

A strain of Saccharomyces cerevisiae lacking the GPD2 gene, encoding one of the glycerol-3-phosphate dehydrogenases, grows slowly under anaerobic conditions, due to reductive stress caused by the accumulation of cytoplasmic NADH. We used 2D-PAGE to study the effect on global protein expression of reductive stress in the anaerobically grown gpd2 strain. The most striking response was a strongly elevated expression of Tdh1p, the minor isoform of glyceraldehyde-3-phosphate dehydrogenase. This increased expression could be reversed by the addition of acetoin, a NADH-specific redox sink, which furthermore largely restored anaerobic growth of the gpd2 strain. Additional deletion of the TDH1 gene (but not of TDH2 or TDH3) improved anaerobic growth of the gpd2 strain. We therefore propose that TDH1 has properties not displayed by the other TDH isogenes and that its expression is regulated by reductive stress caused by an excess of cytoplasmic NADH.H. Valadi and Å. Valadi contributed equally to this work     

Bleomycin-kanamycin resistance as a marker of the presence of transposon Tn5 in clinical strains ofEscherichia coli

The aminoglycoside modifying enzyme aminoglycoside 3- phosphotransferase II (APH(3)II) is encoded for on transposon Tn5 by theaphA gene, in the same operon as theble gene determining bleomycin resistance. To document this linkage 82 kanamycin-resistantEscherichia coli strains of clinical origin were studied; all 18 isolates presenting bleomycin-kanamycin resistance were shown by an enzymatic assay to produce APH(3)II, and the presence of Tn5 was demonstrated by gene hybridization. Similarly, bleomycin-kanamycin resistance was shown to be linked to APH(3)II production inSalmonella spp. The epidemiology of strains with Tn5-encoded APH(3)II may thus be studied, at least inEscherichia coli, by a simple diffusion test using bleomycin and kanamycin discs.     

Expression of myosin heavy and light chains and phosphorylation of the phosphorylatable myosin light chain in the heart ventricle of the European hamster during hibernation and in summer

Summary We investigated the expression of myosin subunits (myosin heavy chains) as well as light chains and thein vivo phosphorylation of the phosphorylatable myosin light chain in the heart ventricle of the adult male European hamster (Cricetus cricetus L.). Two myosin heavy chain isoenzymes could be detected under native and denaturing electrophoretic conditions having high (-myosin heavy chain) and low (-myosin heavy chain) enzymatic activity. Enzymatic activity of- and-myosin heavy chain revealed a different temperature dependency. When temperature increased ATPase activity of the-myosin heavy chain isoenzyme increased relatively more than ATPase activity of the-myosin heavy chain isoenzyme. Summer animals expressed predominantly the-myosin heavy chain (79% of total myosin) while during hibernation the-myosin heavy chain expression increased to 53% of total myosin. Winter-active hamsters kept at 22°C and 12 h day/night rhythm showed the same myosin heavy chain isoenzyme pattern as summer-active animals. Two myosin light chain forms were expressed in the ventricle of all animal groups. thein vivo phosphorylation level of the phosphorylatable myosin light chain decreased from 45% in summer-active hamster to 23% during hibernation.     

Modulation of hematopoietic colony formation of stem cells in peripheral blood by anti-TGF-β in patients with severe immunosuppression

Summary The influence of transforming growth factor- (TGF-) on hematopoiesis has been evaluated by adding blocking antibodies against TGF- to colony forming assays (CFU-c). When optimum concentrations of recombinant growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-3 (IL-3) were added to stem cells from the peripheral blood of healthy individuals and certain patients with tumors or HIV infection, the anti-TGF- capable of blocking 5 ng/ml of active TGF- had no significant influence on erythroid or myeloid colony formation. However, in certain immunosuppressed individuals, anti-TGF- resulted in a significant decrease of erythroid colony formation and slight suppression of myeloid colony formation. The significant inhibition of hematopoiesis by plasma of HIV patients could be due to the presence of active forms of TGF-. The results of the blocking experiments are consistent with the concept that TGF- in low concentrations is essential for erythropoiesis and myelopoiesis but that higher levels of TGF- primarily inhibit erythropoiesis in vitro. TGF- serves as a coordinating factor when efficient recruitment of granulocytes and monocytes is more essential than erythropoiesis and stem cell growth.Abbreviations BFU-E burst forming unit-erythroid - CFC colony forming cells - CFU-GEMM colony forming unit-granulocyte/erythroid/macrophage/megacaryocyte - CFU-GM colony forming unit-granulocyte/macrophage - EPO erythropoietin - GM-CSF granulocyte/macrophage-colony stimulating factor - HIV human immunodeficiency virus - IL-1 interleukin-1 - IL-3 interleukin-3 - IMDM Iscove's Modified Dulbecco's medium - PBS phosphate buffered saline - TGF- transforming growth factor- - TNF- tumor necrosis factor-     

Comparison of expression of the endo-β-1,3-1,4-glucanase gene from Bacillus subtilis in Saccharomyces cerevisiae from the CYC1 and ADH1 promoters

Summary The endo--1,3-1,4-glucanase gene from B. subtilis was placed under yeast promoter control in a number of different yeast expression vectors. The hybrid plasmids were transformed into S. cerevisiae where they directed the synthesis of varying amounts of active enzyme. The presence of B. subtilis DNA sequences 5 to the initiation codon for the B. subtilis -glucanase gene reduced expression of the gene in yeast. A 1,000-fold increase in the yield of -glucanase was obtained using the ADH1 promoter compared with the CYC1 promoter.     

A method for easy isolation of promoter fragments from promoter-probe libraries of filamentous fungi

Two genomic fragments capable of driving the expression of the hygromycin B resistance gene (hph) were isolated from the phytopathogenic ascomycete Gibberella pulicaris (anamorph Fusarium sambucinum) using a promoter-probe library strategy. Two libraries consisting of random, 0.5–2.0-kb fragments of genomic DNA inserted 5 of a promoterless hph gene were constructed and used for transformation of G. pulicaris. Both libraries transformed G. pulicaris at a low frequency. Transformants tolerated up to 800 g/ml of hygromycin B, while untransformed colonies were inhibited completely by 50 g/ml of the antibiotic. Plasmids were re-isolated from transformants by simply digesting, the genomic DNA with KpnI, which cuts once in the polylinker 5 to the insert, and transforming E. coli with the re-ligated DNA. The recovered plasmids transformed G. pulicaris with a frequency of up to 4.4 transformants/g of DNA. Both promoter fragments were sequenced and found to contain TATA and CAAT boxes as well as CT-rich sequences. This method makes it possible to easily isolate many fragments with promoter activity from filamentous fungi, and should facilitate the investigation of the promoter structures necessary for the expression of fungal genes.     

Nucleotide sequence of a Singapore isolate of zucchini yellow mosaic virus coat protein gene revealed an altered DAG motif

A cDNA clone of zucchini yellow mosaic virus (ZYMV) RNA was mapped to the 3 terminal region. The nucleotide sequence revealed a single open reading frame of 1035 nucleotides followed by a 3 noncoding region of 215 nucleotides. The putative protease cleavage site for the release of coat protein (CP) was deduced to be between Glu-Ser (at amino acid position 66–67), which would result in a protein of 279 amino acids. This non-aphid-transmissible Singapore isolate of ZYMV showed a change of DAG to GAG triplet near the N-terminal of the CP. The CP gene was expressed as a protein fused to the -galactosidase inEscherichia coli and as an unfused protein inSaccharomyces cerevisiae.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number X62662.     

A homologous and self-replicating system for efficient transformation ofFusarium oxysporum

A highly efficient transformation system has been developed forFusarium oxysporum f. sp.lycopersici based on the complementation of a nitrate-reductase mutant with the homologousnitI gene and on the presence ofARS and telomeric sequences in the vector. Preliminary transformation experiments with theniaD gene fromAspergillus niger generated self-replicating plasmids within the transformed entity that contained extra-fungal DNA. A fragment of the extra DNA was inserted into pUC19 together with theF. oxysporum nitl gene, resulting in plasmid pFNit-Lam. This allowed the isolation of a new linear plasmid within self-replicativeF. oxysporum transformants (pFNit-Lam-TLam, linear). The circular form of this vector yielded 5600 fungal transformants per g of DNA. All of the transformants contained autonomous linear plasmids harboring direct repeats of fungal DNA at both ends. The sequence of the 1.2-kb fragment fromF. oxysporum responsible for autonomous replication, and maintenance as linear plasmid molecules, has been determined. Comparison analysis with theARS from different organisms has shown that this fragment contained the commonly identifiedARS consensus sequence, 5A/TTTTATA/GTTTA/T3 and, in addition to this core, ten copies of theARS-box, TNTA/GAA3. Adjacent to this presumedARS, the telomeric hexanucleotide sequence (TTAGGG)_n was present in six tandem copies followed by 18 copies of its complementary sequence.     

Effect of theophylline onβ-adrenergic receptor density and cAMP content in bovine aortic smooth muscle cells

Activation of vascular-adrenergic receptors prevents an increase in vascular permeability caused by free radicals or inflammatory peptides. Methylxanthines seem to have similar protective effects on vascular endothelium. In the present study we investigated the effect of theophylline on the-adrenergic receptor expression and cAMP concentrations in cultured endothelial and smooth muscle cells from bovine aorta. Comparable values for-receptor density and binding affinity were detected in both cell types. Isoproterenol induced significant downregulation of-receptors in endothelial (BAEC: –60.5%) and smooth muscle cells (BASMC: –52.5%; P < 0.01). Incubation of endothelial cells with theophylline (4 µg/ml and 40 µg/ml) for 24 hours did not affect-receptor expression, whereas in smooth muscle cells the-receptor density was reduced for –31.5% and –28.7, respectively. In endothelial cells a transient effect on cAMP concentrations was observed after stimulation with isoproterenol (1 µM), but no effect was found in theophylline treated endothelial cells. Stimulation of intact smooth muscle cells with isoproterenol and theophylline (4 µg/ml and 40 µg/ml) resulted in a significant increase of cAMP concentrations after 60 and 240 minutes. The present data suggest a novel, celltype specific effect of theophylline on the-adrenergic receptor expression in vascular smooth muscle cells in vitro.     

Chloroplast RNA polymerase genes of Chlamydomonas reinhardtii exhibit an unusual structure and arrangement

Summary Nucleotide sequence analysis of a 17043 basepair (bp) region of the Chlamydomonas reinhardtii plastome indicates the presence of three open reading frames (ORFs) similar to RNA polymerase subunit genes. Two, termed rpoB1 and rpoB2, are homologous to the 5-and 3-halves of the Escherichia coli beta subunit gene, respectively. A third, termed rpoC2, is similar to the 3-half of the bacterial beta' subunit gene. These genes exhibit several unusual features: (1) all three represent chimeric structures in which RNA polymerase gene sequences are juxtaposed in-frame with long sequences of unknown identity; (2) unlike their counterparts in plants and eubacteria, rpoB1 and rpoB2 are separated from rpoC2 by a long (7 kilobase-pair, kbp) region containing genes unrelated to RNA polymerase; (3) DNA homologous to the 5 half of rpoC (termed rpoC1 in other species) is not present at the 5 end of rpoC2 and could not be detected in C. reinhardtii chloroplast DNA. RNA expression could not be detected for any of the RNA polymerase genes, suggesting that they are pseudogenes or genes expressed at stages of the C. reinhardtii life-cycle not investigated. The three genes are flanked by GC-rich repeat elements. We suggest that repeat DNA-mediated chloroplast recombination events may have contributed to their unusual arrangement.     

The role ofγδ T cells in the normal and disordered immune system

Summary A small population of T cells does not express the conventional T cell receptor characterized by the and polypeptide chains (TCR) but instead, two polypeptides termed and (TCR). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR remains unclear. In selected sites such as skin or gut TCR predominates in mice which might suggest a role of T cells in the first line of defense against infection, T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for T cells since MHC-nonrestricted and also CD4 and CD8 negative T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the T cells. In various diseases slightly increased numbers of T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of T cells.List of abbreviations C constant region (immunoglobulin or TCR gene segment) - CD4 cluster of differentiation 4 (mainly on helper cells) - CD8 cluster of differentiation 8 (mainly on cytotoxic cells) - D diversity region (immunoglobulin or TCR gene segment) - DNA desoxyribonucleic acid - IL2 interleukin 2 - J joining region (immunoglobulin or TCR gene segment) - kD kiloDalton - MHC major histocompatibility complex - NK natural killer (cells) - RA rheumatoid arthritis - TCR T cell receptor - V variable region (immunoglobulin or TCR gene segment)     

Isolation of the Bα and Bβ mating-type loci of Schizophyllum commune

The B mating type of the basidiomycete fungus, Schizophyllum commune is determined by two, tightly linked, multi-specificity (also called multi-allelic) loci: B and B. A plasmid library was used in DNA-mediated transformation to obtain transformants that displayed B-directed development. Plasmids that conferred B1 and B1 mating-type specificities were rescued from the transformants. Fragments of DNA from each plasmid hybridized to genomic DNA from the strain used to make the plasmid library; however, they did not hybridize, or hybridized only weakly, to genomic DNA from strains with mating-type specificities different from B1 or B1. The cloned fragments are presumed to correspond to active regions of each B mating-type locus.     

β subunits determine the time course of desensitization in rat α3 neuronal nicotinic acetylcholine receptors

Standard two-electrode voltage-clamp techniques were used to investigate some of the pharmacological and functional properties of two types of rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes after pairwise injection of 34 or 32 mRNAs. Currents of several A amplitude were elicited by fast application of micromolar concentrations of either acetylcholine (ACh) or 1,1-dimethyl-4-piperazine (DMPP). The activation of either receptor type by DMPP showed cooperativity (Hill coefficient, n1.7) with a half-maximal activation concentration (EC₅₀) of 15–30 M. In 34 receptors, ACh displayed cooperativity (n=1.8) but was less efficacious than DMPP, yet its EC₅₀ was about equal to that of DMPP. Finally, in 32 receptors, ACh was much less efficacious and had a much lower EC₅₀. Desensitization induced by either DMPP or ACh was slow in 34 nicotinic ACh receptors but was rapid and extensive in 32 receptors, causing a significant proportion of the response to wane within the first few seconds of agonist application.