A two unit antisense RNA cassette test system for silencing of target genes

This communication describes a two unit antisense RNA cassette system for use in gene silencing. Cassettes consist of a recognition unit and an inhibitory unit which are transcribed into a single RNA that carries sequences of non-contiguous complementarity to the chosen target RNA. The recognition unit is designed as a stem-loop for rapid formation of long- lived binding intermediates with target sequences and resembles the major stem-loop of a naturally occurring antisense RNA, CopA. The inhibitory unit consists of either a sequence complementary to a ribosome binding site or of a hairpin ribozyme targeted at a site within the chosen mRNA. The contributions of the individual units to inhibition was assessed using the lacI gene as a target. All possible combinations of recognition and inhibitory units were tested in either orientation. In general, inhibition of lacI expression was relatively low. Fifty per cent inhibition was obtained with the most effective of the constructs, carrying the recognition stem-loop in the antisense orientation and the inhibitory unit with an anti-RBS sequence. Several experiments were performed to assess activities of the RNAs in vitro and in vivo : antisense RNA binding assays, cleavage assays, secondary structure analysis as well as Northern blotting and primer extension analysis of antisense and target RNAs. The problems associated with this antisense RNA approach as well as its potential are discussed with respect to possible optimization strategies.     

A shuttle mutagenesis system for tagging genes in the yeast Yarrowia lipolytica

A high-performance liquid chromatography (HPLC) method to determine the most important cellular thiols [reduced glutathione (GSH), cysteine, gamma-glutamylcysteine and cysteinylglycine] is described. Separation relies upon isocratic ion-pairing reversed-phase chromatography and detection is operated by spectrofluorimetry coupled with post-column derivatization reactions using either N-(1-pyrenyl)maleimide (NPM) or ortho-phthalaldehyde (OPA). When OPA is used without co-reagent, only GSH and gamma-glutamylcysteine are detected (heterobifunctional reaction). However, either the OPA reaction in the presence of glycine in the mobile phase (thiol-selective reaction) or NPM allows the detection of all the cited thiols. The HPLC system has been validated as concerning linearity, accuracy and precision. The low detection limits reached (in the pmol range for each thiol injected) allow the screening and the quantification of thiols (as NPM derivatives) in V79cl and V79HGGT cells as well as the measurement of two cytosolic enzymes related to the glutathione synthesis, using the heterobifunctional OPA reaction.     

A catalogue of genes in the cardiovascular system as identified by expressed sequence tags

The heart, which is composed of all the cellular components of the circulatory system, is a representative organ for obtaining genes expressed in the cardiovascular system in normal and disease states. We used partial sequences of cDNA clones, or expressed sequence tags, to identify and tag genes expressed in this organ. More than 3500 partial sequences representing > 3000 cDNA clones have been obtained from either the 5' or 3' end of inserts derived from human heart cDNA libraries. Of 3132 cDNA clones analyzed by sequence similarity searching against the GenBank/EMBL data bases, 1485 (47.4%) were found to represent additional, previously undiscovered genes, whereas 267 clones were matched to human brain expressed sequence tags. Clones matching to known genes were catalogued according to their putative structural and cellular functions. cDNA probes from reverse-transcribed mRNAs of fetal and adult hearts were used to study differential expression of selected clones in cardiac development. Cataloguing genes expressed in the heart may provide insight into the genes involved in health and cardiovascular disease.     

Molecular population genetics of Drosophila immune system genes

A striking aspect of many vertebrate immune system is the exceptionally high level of polymorphism they harbor. A convincing case can be made that this polymorphism is driven by the diversity of pathogens that face selective pressures to evade attack by the host immune system. Different organisms accomplish a defense against diverse pathogens through mechanisms that differ widely in their requirements for specific recognition. It has recently been shown that innate defense mechanisms, which use proteins with broad-spectrum bactericidal properties, are common to both primitive and advanced organisms. In this study we characterize DNA sequence variation in six pathogen defense genes of Drosophila melanogaster and D. mauritiana, including Andropin; cecropin genes CecA1, CecA2, CecB, and CecC; and Diptericin. The necessity for protection against diverse pathogens, which themselves may evolve resistance to insect defenses, motivates a population-level analysis. Estimates of variation levels show that the genes are not exceptionally polymorphic, but Andropin and Diptericin have patterns of variation that differ significantly from neutrality. Patterns of interpopulation and interspecific differentiation also reveal differences among the genes in evolutionary forces.     

Selection system for genes encoding nuclear-targeted proteins

Nuclear proteins have essential roles in cell proliferation and differentiation. We have developed a yeast selection system-the nuclear transportation trap (NTT)-to identify genes encoding nuclear transport signals. Both unknown and previously identified nuclear localization signals were identified from a human fetal brain cDNA library. The majority (75%) of the unknown proteins examined were exclusively localized to the nucleus in COS-7 cells. We propose that NTT is an efficient method for isolating cDNAs that encode nuclear targeted proteins that can be applied to the retrieval of novel nuclear proteins and to annotate gene function.     

Expression of interleukin-1 system genes in human gametes

Elevation of extracellular calcium has been shown to inhibit osteoclastic bone resorption and stimulate proliferation and chemotaxis of osteoblasts. Therefore, calcium released by bone resorption may have important roles in the coupling of bone resorption and bone formation. Although both osteoclasts and osteoblasts have calcium-sensing mechanisms, the responsible molecule in these cells seems to be different. Functional and histological studies show that calcium-sensing mechanism in osteoclasts is a ryanodine receptor-like molecule in plasma membrane. In contrast, calcium-sensing mechanism in osteoblasts has similar functional property to parathyroid calcium-sensing receptor (CaSR), but is a different molecule from CaSR. In addition, several bone marrow cells express CaSR. The elucidation of the identity and the physiological roles of these calcium-sensing mechanisms would give us a more clear view of bone remodeling.     

Variants of renin-angiotensin system genes and echocardiographic left ventricular mass

AIMS: Variants of renin-angiotensin system genes are shown to be associated with cardiovascular pathology. The association between renin-angiotensin system genes and left ventricular mass was investigated in a population-based case-control study. METHODS AND RESULTS: The association between echocardiographic left ventricular mass and both insertion/deletion polymorphism of the angiotensin-converting enzyme gene and the methionine-threonine variant at position 235 of the angiotensinogen gene was studied in a random cohort of 430 hypertensive and 426 control subjects. No differences in the adjusted left ventricular mass values between the different genotypes were seen among either the hypertensive or the control subjects, whether men or women, or in the subgroups of normotensive or physically active subjects. Gene variation had no statistically significant synergistic effect on left ventricular mass values. In control women, the deletion allele of the angiotensin-converting enzyme gene was associated with an increased risk of left ventricular hypertrophy. However, this finding was based on a small number of women with left ventricular hypertrophy and should be interpreted with caution. CONCLUSION: Variations in renin-angiotensin system genes had no major effect on left ventricular mass in this middle-aged population-based cohort of hypertensives and control subjects.     

Cloning and characterization of the genes of the CeqI restriction-modification system

Two genes from Corynebacterium equii, a Gram-positive bacterium producing the CeqI restriction-modification enzymes were cloned and sequenced. In vivo restriction experiments, DNA and amino acid sequence data suggest that the two genes code for the endonuclease and the methyltransferase enzymes. However, when the two genes are expressed in E. coli, practically no enzyme activity can be detected in the supernatants of sonicated cells. Based on the DNA sequence data CeqI restriction endonuclease (an EcoRV izoschizomer) consists of 270 amino acid residues with a predicted molecular mass of 31.6 kDa, in good agreement with the previously measured 32 +/- 2 kDa. The methyltransferase is 517 residues long (approx. 60 kDa). The two genes are in opposite orientation and overlap by 37 base pairs on the chromosome. The deduced amino acid sequence of the putative endonuclease gene revealed long stretches of hydrophobic amino acids, that may form the structural basis of the unusual aggregation properties of the restriction endonuclease. The amino acid sequence of the methylase shows homologies with other type II methyltransferases.     

Novel phosphotransferase system genes revealed by bacterial genome analysis: the complete complement of pts genes in mycoplasma genitalium

The complete sequence of the Mycoplasma genitalium chromosome has recently been determined. We here report analyses of the genes encoding proteins of the phosphoenolpyruvate:sugar phosphotransferase system, PTS. These genes encode (1) Enzyme I, (2) HPr, (3) a glucose-specific Enzyme IICBA, (4) an inactive glucose-specific Enzyme IIB, lacking the active site cysteyl residue, and (5) a fructose-specific Enzyme IIABC. Some of the unique features of these genes and their enzyme products are as follows. (1) Each of the genes is encoded within a distinct operon. (2) Both Enzyme I and HPr have basic isoelectric points. (3) The glucose-specific Enzyme IIC bears a centrally located, hydrophilic, 200 amino acyl residue insert that lacks sequence similarity with any protein in the current database. (4) The fructose-specific Enzyme II has a domain order (IIABC), different from those of previously characterized fructose permeases, and its IIA domain more closely resembles the IIANtr protein of Escherichia coli than other fructose-specific IIA domains. The potential significance of these novel features is discussed.     

A new class of Caulobacter crescentus flagellar genes

Eight Caulobacter crescentus flagellar genes, flmA, flmB, flmC, flmD, flmE, flmF, flmG, and flmH, have been cloned and characterized. These eight genes are clustered in pairs (flmAB, flmCD, flmEF, and flmGH) that appear to be structurally organized as operons. Homology comparisons suggest that the proteins encoded by the flm genes may be involved in posttranslational modification of flagellins or proteins that interact with flagellin monomers prior to their assembly into a flagellar filament. Expression of the flmAB, flmEF, and flmGH operons was shown to occur primarily in predivisional cells. In contrast, the flmCD operon was expressed throughout the cell cycle, with only a twofold increase in predivisional cells. The expression of the three temporally regulated operons was subject to positive regulation by the CtrA response regulator protein. Mutations in class II and III flagellar genes had no significant effect on the expression of the flm genes. Furthermore, the flm genes did not affect the expression of class II or class III flagellar genes. However, mutations in the flm genes did result in reduced synthesis of the class IV flagellin proteins. Taken together, these data indicate that the flm operons belong to a new class of flagellar genes.     

A genome-wide search for schizophrenia susceptibility genes

We completed a systematic genome-wide search for evidence of loci linked to schizophrenia using a collection of 70 pedigrees containing multiple affected individuals according to three phenotype classifications: schizophrenia only (48 pedigrees; 70 sib-pairs); schizophrenia plus schizoaffective disorder (70 pedigrees; 101 sib-pairs); and a broad category consisting of schizophrenia, schizoaffective disorder, paranoid or schizotypal personality disorder, psychosis not otherwise specified (NOS), delusional disorder, and brief reactive psychosis (70 pedigrees; 111 sib-pairs). All 70 families contained at least one individual affected with chronic schizophrenia according to DSM-III-R criteria. Three hundred and thirty-eight markers spanning the genome were typed in all pedigrees for an average resolution of 10.5 cM (range, 0-31 cM) and an average heterozygosity of 74.3% per marker. The data were analyzed using multipoint nonparametric allele-sharing and traditional two-point lod score analyses using dominant and recessive, affecteds-only models. Twelve chromosomes (1, 2, 4, 5, 8, 10, 11, 12, 13, 14, 16, and 22) had at least one region with a nominal P value <0.05, and two of these chromosomes had a nominal P value <0.01 (chromosomes 13 and 16), using allele-sharing tests in GENEHUNTER. Five chromosomes (1, 2, 4, 11, and 13) had at least one marker with a lod score >2.0, allowing for heterogeneity. These regions will be saturated with additional markers and investigated in a new, larger set of families to test for replication.     

GeneDn: for high-level expression design of heterologous genes in a prokaryotic system

RESULTS: Based on the mathematical model of high-level expression of heterologous genes in prokaryotic vector pBV220, we developed a program GeneDn for high-level expression design of natural and synthetic genes. AVAILIBILITY: The program is written in Turbo Pascal 7.0. The source code and related material are available upon request. CONTACT: wujj@nic.bmi.ac.cn     

A transgenic mouse strain expressing four drug-selectable marker genes

Murine embryonic stem (ES) cells are commonly cultured on feeder layers of primary murine embryonic fibroblasts (MEFs). Because gene targeting experiments often involve sequential selection for multiple-drug resistance in single ES cell lines, we have developed a new mouse strain which represents an economical donor for the production of multiple-drug resistant MEFs. MEFs prepared from the DR-4 mouse strain displayed resistance to concentrations of the drugs G418, 6-thioguanine, puromycin and hygromycin well above those used normally for the selection of drug-resistant ES cells.     

Familial correlations and X-linked genes: A comment.

Presents a simpler alternative to H. Thomas's (see record 1983-25050-001) formula for the phenotypic correlation among family members when a trait is affected by an X-linked gene. The present author suggests that the ordering of phenotypic familial correlations may not be entirely hopeless as a source of evidence for X linkage. (4 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)