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PubMed1999

An efficient and accurate integration of mini-Mu transposons in vitro: a general methodology for functional genetic analysis and molecular biology applications.

Transposons are mobile genetic elements and have been utilized as essential tools in genetics over the years. Though highly useful, many of the current transposon-based applications suffer from various limitations, the most notable of which are: (i) transposition is performed in vivo, typically species specifically, and as a multistep process; (ii) accuracy and/or efficiency of the in vivo or in vitro transposition reaction is not optimal; (iii) a limited set of target sites is used. We describe here a genetic analysis methodology that is based on bacteriophage Mu DNA transposition and circumvents such limitations. The Mu transposon tool is composed of only a few components and utilizes a highly efficient and accurate in vitro DNA transposition reaction with a low stringency of target preference. The utility of the Mu system in functional genetic analysis is demonstrated using restriction analysis and genetic footprinting strategies. The Mu methodology is readily applicable in a variety of current and emerging transposon-based techniques and is expected to generate novel approaches to functional analysis of genes, genomes and proteins.

Base Sequence↗
PubMed1988

Correlation of physical maps and some genetic functions in the genomes of the kappa-theta phage family of Bacillus licheniformis.

Natural recombinant genomes between several, phenotypically distinct forms of phages kappa and theta were isolated and characterized by DNA restriction fragment mapping and electron microscopic heteroduplex analysis. The phenotypes of the recombinants were correlated with the physical maps of the genomes, and several genetic functions were therefore defined and mapped. All genes necessary for the assembly of infectious virus particles map in a contiguous tract of DNA comprising about 20 kb, or nearly one third of the genome length. No DNA homology occurs within these domains of the two genomes, so that homologous recombination does not take place here and phenotypic mixing of the phages is eo ipso excluded. Other regions of heterology contain regulatory genes responsible for the lytic or temperature character of the phages, and for exclusion of phage kappa by 9.

Bacillus↗
PubMed2002

Information overload: assigning genetic functionality in the age of genomics and large-scale screening.

As more and more genome sequences are completed, it is becoming increasingly evident that our understanding of the function of most bacterial gene products is lacking. This is frustrating, particularly in the study of pathogens, where an understanding of the role of individual gene products would probably facilitate the development of novel antimicrobials and vaccines. Recently, we devised a technique known as virulence-attenuated pool (VAP) screening to help assign genetic functionality to gene products that the pathogen Vibrio cholerae requires for colonization. This screen and potential new applications of the VAP technique are discussed here.

Bacteria↗
PubMed2007

QSAR studies on chalcones and flavonoids as anti-tuberculosis agents using genetic function approximation (GFA) method.

Design of compounds having good anti-tubercular activity is gaining much importance in the field of tuberculosis research due to reemergence of antibiotic resistance strains. In this paper quantitative structure activity relationships (QSAR) were developed on chalcones, chalcone-like compounds, flavones and flavanones to understand the relationship between biological activity and structural features. Genetic function approximation (GFA) method was used to identify the descriptors that would lead to good regression equations. The best molecular descriptors identified were Jurs descriptors (Jurs charged partial surface area), hydrogen bond donor, principal moment of inertia, molecular energy, dipole magnetic, molecular area, absorption, distribution, metabolism and excretion (ADME) properties and Chi indices (Kier & Hall chi connectivity indices). Excellent statistically significant models were developed by this approach (r(2)=0.8-0.97) for the four groups of compounds. The cross validated r(2) (XV r(2)) which is an indication of the predictive capability of the model for all the cases was also very good (=0.79-0.94).

Antitubercular Agents↗
PubMed1998

Functional genetic tests of DNA mismatch repair protein activity in Saccharomyces cerevisiae.

Hereditary non-polyposis colorectal cancer (HNPCC) is associated with mutations in four different genes encoding proteins involved in DNA mismatch repair (DMR). As many as 30% of the observed sequence variations in human DMR genes predict minor alterations in the encoded protein, such as amino acid (aa) replacements or small in-frame deletions/insertions. For such sequence variants, a functional genetic test will be required to discriminate mutations from polymorphisms. We have constructed a series of isogenic yeast strains in which individual genes involved in DMR are disrupted, and have standardized an assay which measures GT tract stability (Strand et al., 1993) to characterize these gene products. Disruptions of the yeast MSH2, MLH1, and PMS1 genes result in, respectively, a 290-, 450- and 390-fold increased tract instability over the wild type (wt) strain under optimized assay conditions. Expression of the wt MSH2 and PMS1 gene from plasmids results in complementation of the corresponding chromosomal gene disruption. Two different aa replacements which correspond to previously observed sequence variants of the human MSH2 gene, and implicated in HNPCC, were created in the conserved aa of the yeast MSH2 gene by site directed mutagenesis. Conversion of the Pro640 in the yeast protein to Leu resulted in a complete loss of protein function. In contrast, a yeast MSH2p protein in which the His658 is changed to Tyr retains full function in this in vivo assay. These results indicate that the Pro-->Leu and His-->Tyr variants observed in humans constitute, respectively, a mutation and a polymorphism. The system described here may be used for further structure/function analysis of yeast DMR proteins. Such studies may provide insight into the effects that specific sequence variations observed in human DMR proteins have on their function.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Functional genetic analysis of mutations implicated in a human speech and language disorder.

Mutations in the FOXP2 gene cause a severe communication disorder involving speech deficits (developmental verbal dyspraxia), accompanied by wide-ranging impairments in expressive and receptive language. The protein encoded by FOXP2 belongs to a divergent subgroup of forkhead-box transcription factors, with a distinctive DNA-binding domain and motifs that mediate hetero- and homodimerization. Here we report the first direct functional genetic investigation of missense and nonsense mutations in FOXP2 using human cell-lines, including a well-established neuronal model system. We focused on three unusual FOXP2 coding variants, uniquely identified in cases of verbal dyspraxia, assessing expression, subcellular localization, DNA-binding and transactivation properties. Analysis of the R553H forkhead-box substitution, found in all affected members of a large three-generation family, indicated that it severely affects FOXP2 function, chiefly by disrupting nuclear localization and DNA-binding properties. The R328X truncation mutation, segregating with speech/language disorder in a second family, yields an unstable, predominantly cytoplasmic product that lacks transactivation capacity. A third coding variant (Q17L) observed in a single affected child did not have any detectable functional effect in the present study. In addition, we used the same systems to explore the properties of different isoforms of FOXP2, resulting from alternative splicing in human brain. Notably, one such isoform, FOXP2.10+, contains dimerization domains, but no DNA-binding domain, and displayed increased cytoplasmic localization, coupled with aggresome formation. We hypothesize that expression of alternative isoforms of FOXP2 may provide mechanisms for post-translational regulation of transcription factor function.

Alternative Splicing↗
PubMed1995

Genetic functions of isolated maize mitochondria under model changes of redox conditions.

The template functions of maize mitochondrial genome have been studied under model conditions of redox potential created by the addition of potassium ferricyanide as an oxidising agent and sodium dithionite as a reducing agent. The addition of both potassium ferricyanide and sodium dithionite to the isolated mitochondria causes the changes in the activity of the mitochondria RNA and DNA synthesis. The data obtained indicate the possibility of redox regulation of genetic functions in isolated mitochondria.

Alkaloids↗
PubMed2004

An in vivo functional genetic screen reveals a role for the TRK-T3 oncogene in tumor progression.

Over the past decades, much has been learnt about the genes that contribute to oncogenic transformation of primary cells in vitro. However, much less is known about the genes that contribute to the later stages of tumor progression, in which cells of ever increasing malignancy arise through clonal selection in vivo. To search for genes that confer a tumor progression phenotype in vivo, we have used a functional genetic approach. We used adenovirus-transformed mouse embryo fibroblasts, which are tumorigenic in immunodeficient nude mice, but not in immunocompetent mice, due to strong cytotoxic T-cell-mediated immune rejection. We infected these cells in vitro with several high-complexity retroviral cDNA expression libraries and selected rare variants that formed tumors in immunocompetent mice. Using this approach, we identify here the TRK-T3 oncogene as a tumor progression gene. TRK-T3 does not inhibit T-cell reactivity towards the tumor cells. Instead, we find that cells expressing TRK-T3 enhances in vivo growth rate, most likely by stimulating anchorage-independent proliferation in growth factor-limiting conditions. Our data indicate that cDNA expression libraries can be used to identify tumor progression genes in vivo that cannot be readily identified using in vitro cell culture systems.

Animals↗
PubMed1990

Genetic functions and cell-cell interactions in the pheromone-inducible plasmid transfer system of Enterococcus faecalis.

Pheromone-inducible plasmid transfer is a novel form of bacterial conjugation which has, to date, been observed only in Enterococcus (Streptococcus) faecalis. This process includes several important stages of interaction between the donor and recipient cell. The initial interaction is the transmission of a chemical signal from the recipient to the donor cell. Recent evidence has shown that the signal is in the form of a small hydrophobic peptide, which is capable of inducing a complex mating response in the donor cell at concentrations as low as 1-5 molecules per responder cell. Most E. faecalis strains produce multiple pheromones, each of which induces a response only in cells carrying a particular plasmid (or member of a family of related plasmids). Genetic functions ascribed to the pheromone response include: (i) cell-cell aggregation, which promotes initial close contact between mating cells; (ii) surface exclusion, which prevents plasmid transfer between aggregated donor cells; and (iii) highly efficient DNA transfer, which requires other unidentified functions in addition to aggregation. The first two processes appear to be mediated by proteinaceous surface antigens.

Amino Acid Sequence↗
PubMed2001

Complementation of the Magnaporthe grisea deltacpkA mutation by the Blumeria graminis PKA-c gene: functional genetic analysis of an obligate plant pathogen.

Obligate plant-pathogenic fungi have proved extremely difficult to characterize with molecular genetics because they cannot be cultured away from host plants and only can be manipulated experimentally in limited circumstances. Previously, in order to characterize signal transduction processes during infection-related development of the powdery mildew fungus Blumeria graminis (syn. Erysiphe graminis) f. sp. hordei, we described a gene similar to the catalytic subunit of cyclic AMP-dependent protein kinase A (here renamed Bka1). Functional characterization of this gene has been achieved by expression in a deltacpkA mutant of the nonobligate pathogen Magnaporthe grisea. This nonpathogenic M. grisea deltacpkA mutant displays delayed and incomplete appressorium development, suggesting a role for PKA-c in the signal transduction processes that control the maturation of infection cells. Transformation of the deltacpkA mutant with the mildew Bka1 open reading frame, controlled by the M. grisea MPG1 promoter, restored pathogenicity and appressorium maturation kinetics. The results provide, to our knowledge, the first functional genetic analysis of pathogenicity in an obligate pathogen and highlight the remarkable conservation of signaling components regulating infection-related development in pathogenic fungi.

Archaeal Proteins↗
PubMed2006

Evidence for a functional genetic polymorphism of the human mercaptopyruvate sulfurtransferase (MPST), a cyanide detoxification enzyme.

Mercaptopyruvate sulfurtransferase (MPST) plays a central role in both cysteine degradation and cyanide detoxification. Moreover, deficiency in MPST activity has been suggested to be responsible for a rare inheritable disorder known as mercaptolactate-cysteine disulfiduria (MCDU). To date, no mutation of the human MPST gene has been reported. We developed a screening strategy to search for mutations in the MPST gene of 50 unrelated French individuals. Two intronic polymorphisms (IVS1-110C>G and IVS2+39C>T) and a nonsense mutation (Tyr(85)Stop) were identified and their functional consequences were assessed in vivo by measurement of erythrocyte MPST activity and/or in vitro using heterologous expression or transient transfection assay. The nonsense mutation likely leads to the synthesis of a severely truncated protein without enzymatic activity, as supported by our in vitro data. This work constitutes the first report of the existence of a functional genetic polymorphism affecting MPST and should be of great help to investigate certain disorders such as MCDU.

Amino Acid Metabolism, Inborn Errors↗
PubMed2005

QSAR by LFER model of cytotoxicity data of anti-HIV 5-phenyl-1-phenylamino-1H-imidazole derivatives using principal component factor analysis and genetic function approximation.

Cytotoxicity data of anti-HIV 5-phenyl-1-phenylamino-1H-imidazole derivatives were subjected to quantitative structure-activity relationship (QSAR) study using linear free energy related (LFER) model of Hansch using electronic (Hammett sigma), hydrophobicity (pi) and steric (molar refractivity and STERIMOL L, B1, B2, B3 and B4) parameters of phenyl ring substituents of the compounds, along with appropriate indicator variables. Principal component factor analysis (FA) was used as the data-preprocessing step to identify the important predictor variables contributing to the response variable and to avoid collinearities among them. The generated multiple linear regression (MLR) equations were statistically validated using leave-one-out technique. Genetic function approximation (GFA) was also used on the same data set to develop QSAR equations, which produced the same best equation as obtained with FA-MLR. The final equation is of acceptable statistical quality (explained variance 80.2%) and predictive potential (leave-one-out predicted variance 74%). The analysis explores the structural and physicochemical contributions of the compounds for cytotoxicity. A thiol substituent at 2 position of the imidazole nucleus decreases cytotoxicity when compared to the corresponding unsubstituted congener. Presence of hydrogen bond donor group at meta position of the phenyl ring present at 5 position of the imidazole nucleus also reduces cytotoxicity. Additionally, absence of any substituent at 2 and 3 positions of the phenyl ring of 1-phenylamino fragment reduces the cytotoxicity. The negative coefficient of sigmap indicates that presence of electron-withdrawing substituents at the para position of the phenyl ring of the 1-phenylamino fragment is not favourable for the cytotoxicity. Again, lipophilicity of meta substituents of the 5-phenyl ring increases cytotoxicity. The coefficients of molar refractivity (MRm) and STERIMOL parameters for meta substituents (Lm, B1m and B4m) of the phenyl ring of 1-phenylamino fragment indicate that the length, width and overall size of meta substituents are conducive factors for the cytotoxicity.

Algorithms↗
PubMedDate not supplied

[Role of non-histone proteins in the transformations and genetic functioning of the chromosome].

The functional relationship between the transformations of chromosome structure and parallel changes of its major components, i. e. DNA, histones and non-histone proteins (NHP) has been analysed. These changes of chromosome have been checked in the early development, in the highly differentiated undividing cells, in the mitotic and meiotic cells, till the gamete formation. The analysis performed enable us to propose that chromosomal non-histone proteins play the main role in the transformation of chromosome structure, decreasing its genetic activity. NHPs aged without renewal in the undivided nuclei and chromosomes. These proteins take part in the formation of synaptonemal complex (SC) in the early meiotic prophase during chromosome pairing; SC is subsequently eliminated in the diplotene. Thus mature sperms and eggs are characterized by NHP deficiency. NHPs are synthesized de novo in the chromosomes after fertilization providing genetic function of the chromosomes in the development and differentiation of cell systems in new generation of organisms.

Animals↗
PubMed2006

Evidence for a functional genetic polymorphism of the human thiosulfate sulfurtransferase (Rhodanese), a cyanide and H2S detoxification enzyme.

Rhodanese or thiosulfate sulfurtransferase (TST) is a mitochondrial matrix enzyme that plays roles in cyanide detoxification, the formation of iron-sulfur proteins and the modification of sulfur-containing enzymes. Transsulfuration reaction catalyzed by TST is also involved in H(2)S detoxification. To date, no polymorphism of the human TST gene had been reported. We developed a screening strategy based on a PCR-SSCP method to search for mutations in the 3 exons of TST and their proximal flanking regions. This strategy has been applied to DNA samples from 50 unrelated French individuals of Caucasian origin. Eleven polymorphisms consisting in seven nucleotide substitutions in non-coding regions, two silent mutations and two missense mutations were characterized. The functional consequences of the identified mutations were assessed in vivo by measurement of erythrocyte TST activity and/or in vitro using heterologous expression in Saccharomyces cerevisiae or transient transfection assay in HT29 and Caco-2 cell lines. The P(285)A variant appears to encode a protein with a 50% decrease of in vitro intrinsic clearance compared to the wild-type enzyme. Additionally, the six polymorphisms located upstream the ATG initiation codon are responsible for a significant decrease (ranging from 40% to 73%) in promoter activity of a reporter gene compared to the corresponding wild-type sequence. This work constitutes the first report of the existence of a functional genetic polymorphism affecting TST activity and should be of great help to investigate certain disorders for which impairment of CN(-) or H(2)S detoxification have been suggested to be involved.

Cell Line, Tumor↗
PubMed2004

Prep1.1 has essential genetic functions in hindbrain development and cranial neural crest cell differentiation.

In this study we analysed the function of the Meinox gene prep1.1 during zebrafish development. Meinox proteins form heterotrimeric complexes with Hox and Pbx members, increasing the DNA binding specificity of Hox proteins in vitro and in vivo. However, a role for a specific Meinox protein in the regulation of Hox activity in vivo has not been demonstrated. In situ hybridization showed that prep1.1 is expressed maternally and ubiquitously up to 24 hours post-fertilization (hpf), and restricted to the head from 48 hpf onwards. Morpholino-induced prep1.1 loss-of-function caused significant apoptosis in the CNS. Hindbrain segmentation and patterning was affected severely, as revealed by either loss or defective expression of several hindbrain markers (foxb1.2/mariposa, krox20, pax2.1 and pax6.1), including anteriorly expressed Hox genes (hoxb1a, hoxa2 and hoxb2), the impaired migration of facial nerve motor neurons, and the lack of reticulospinal neurons (RSNs) except Mauthner cells. Furthermore, the heads of prep1.1 morphants lacked all pharyngeal cartilages. This was not caused by the absence of neural crest cells or their impaired migration into the pharyngeal arches, as shown by expression of dlx2 and snail1, but by the inability of these cells to differentiate into chondroblasts. Our results indicate that prep1.1 has a unique genetic function in craniofacial chondrogenesis and, acting as a member of Meinox-Pbc-Hox trimers, it plays an essential role in hindbrain development.

Animals↗
PubMedDate not supplied

Mining the NCI anticancer drug discovery databases: genetic function approximation for the QSAR study of anticancer ellipticine analogues.

The U.S. National Cancer Institute (NCI) conducts a drug discovery program in which approximately 10,000 compounds are screened every year in vitro against a panel of 60 human cancer cell lines from different organs of origin. Since 1990, approximately 63,000 compounds have been tested, and their patterns of activity profiled. Recently, we analyzed the antitumor activity patterns of 112 ellipticine analogues using a hierarchical clustering algorithm. Dramatic coherence between molecular structures and activity patterns was observed qualitatively from the cluster tree. In the present study, we further investigate the quantitative structure-activity relationships (QSAR) of these compounds, in particular with respect to the influence of p53-status and the CNS cell selectivity of the activity patterns. Independent variables (i.e., chemical structural descriptors of the ellipticine analogues) were calculated from the Cerius2 molecular modeling package. Important structural descriptors, including partial atomic charges on the ellipticine ring-forming atoms, were identified by the recently developed genetic function approximation (GFA) method. For our data set, the GFA method gave better correlation and cross-validation results (R2 and CVR2 were usually approximately 0.3 higher) than did classical stepwise linear regression. A procedure for improving the performance of GFA is proposed, and the relative advantages and disadvantages of using GFA for QSAR studies are discussed.

Algorithms↗
PubMed1993

Mouse Hox genetic functions.

Functional analysis of the genetic roles of the mammalian Hox homeobox family has not been facilitated by the analysis of disease-associated or classically derived mutant loci. However, an increasing number of mouse Hox mutations, generated by homologous recombination, have now demonstrated that the Hox genes are key components in regulating the patterning of axial structures.

Animals↗
PubMed1998

Murine Otx1 and Drosophila otd genes share conserved genetic functions required in invertebrate and vertebrate brain development.

Despite the obvious differences in anatomy between invertebrate and vertebrate brains, several genes involved in the development of both brain types belong to the same family and share similarities in expression patterns. Drosophila orthodenticle (otd) and murine Otx genes exemplify this, both in terms of expression patterns and mutant phenotypes. In contrast, sequence comparison of OTD and OTX gene products indicates that homology is restricted to the homeodomain suggesting that protein divergence outside the homeodomain might account for functional differences acquired during brain evolution. In order to gain insight into this possibility, we replaced the murine Otx1 gene with a Drosophila otd cDNA. Strikingly, epilepsy and corticogenesis defects due to the absence of Otx1 were fully rescued in homozygous otd mice. A partial rescue was also observed for the impairments of mesencephalon, eye and lachrymal gland. In contrast, defects of the inner ear were not improved suggesting a vertebrate Otx1-specific function involved in morphogenesis of this structure. Furthermore, otd, like Otx1, was able to cooperate genetically with Otx2 in brain patterning, although with reduced efficiency. These data favour an extended functional conservation between Drosophila otd and murine Otx1 genes and support the idea that conserved genetic functions required in mammalian brain development evolved in a primitive ancestor of both flies and mice.

Animals↗