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PubMed2004

Full-length cDNAs from chicken bursal lymphocytes to facilitate gene function analysis.

A large number of cDNA inserts were sequenced from a high-quality library of chicken bursal lymphocyte cDNAs. Comparisons to public gene databases indicate that the cDNA collection represents more than 2,000 new, full-length transcripts. This resource defines the structure and the coding potential of a large fraction of B-cell specific and housekeeping genes whose function can be analyzed by disruption in the chicken DT40 B-cell line.

Animals↗
PubMed1994

Transposon-induced rearrangements in the duplicated locus ph of Drosophila melanogaster can create new chimeric genes functionally identical to the wild type.

Variation in the number of gene copies can play a major role in changing the coding capacities of eukaryotic genomes. Different mechanisms, such as unequal recombination or transposon-induced chromosome rearrangements, are believed to be responsible for these events. We have used the direct tandem duplication at the complex locus polyhomeotic (ph) of Drosophila melanogaster as a model system to study functional redundancy associated with chromosomal rearrangements, such as duplications or deletions. The locus covers 28.6 kb and comprises two independent units, ph proximal and ph distal, which are not only similar on the molecular level, but appear to be functionally redundant [Dura et al., Cell 51 (1987) 829-839; Deatrick et al., Gene 105 (1991) 185-195]. We present a molecular and phenotypic analysis of two hypomorphic ph mutants, ph2 and ph4, induced during hybrid dysgenesis. Each corresponds to an internal deletion in the ph locus that overlaps both transcription units. We show that the deletions are likely due to a P/M hybrid dysgenesis-induced rearrangement between proximal and distal ph, that created a single new chimerical ph gene. At least one of the breakpoints must be located in a 1247-bp region that is rich in single sequence, and 100% identical between proximal and distal ph. Junction points between units are in the protein-coding regions, but could not be exactly localized on the genomic sequence of either mutant, because of the precise molecular mechanism that caused the deletions. Protein products of the hybrid genes contain the same functional domains as either wild-type (wt) product.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2005

Hmx homeobox gene function in inner ear and nervous system cell-type specification and development.

The Hmx homeobox gene family is comprised of three members in mammals, Hmx1, Hmx2, and Hmx3, which are conserved across the animal kingdom and are part of the larger NKL clustered family of homeobox genes. Expression domains of Hmx genes in distantly related species such as Drosophila and mouse suggest an ancestral function in rostral central nervous system development. During vertebrate evolution, the Hmx genes appear to have been recruited into additional roles in inner ear morphogenesis and specification of vestibular inner ear sensory and supporting cell types. Being derived from a common ancestor, the vertebrate Hmx gene family is thus a strong candidate to investigate functional overlap versus the unique roles played by multiple genes belonging to the same family. The functions of Hmx2 and Hmx3 were investigated via directed gene mutagenesis and the primary regions where Hmx2 and Hmx3 exert their individual functions are consistent with their expression domains, such as the vestibule and uterus. Meanwhile, it is notable that some tissues where both Hmx2 and Hmx3 are extensively expressed were not severely affected in either of the Hmx2 or Hmx3 single mutant mice, suggesting a possible functional overlap existing between these two genes. Compound Hmx2 and Hmx3 double mutant mice showed more severe defects in the inner ear than those displayed by either single knockout. Furthermore, novel abnormalities in the hypothalamic-neuroendocrine system, which were never observed in either of the single mutant mice, confirmed a hypothesis that Hmx2 and Hmx3 also function redundantly to control embryonic development of the central nervous system.

Animals↗
PubMed2005

Hox gene function and interaction in the milkweed bug Oncopeltus fasciatus (Hemiptera).

Studies in genetic model organisms such as Drosophila have demonstrated that the homeotic complex (Hox) genes impart segmental identity during embryogenesis. Comparative studies in a wide range of other insect taxa have shown that the Hox genes are expressed in largely conserved domains along the anterior-posterior body axis, but whether they are performing the same functions in different insects is an open question. Most of the Hox genes have been studied functionally in only a few holometabolous insects that undergo metamorphosis. Thus, it is unclear how the Hox genes are functioning in the majority of direct-developing insects and other arthropods. To address this question, we used a combination of RNAi and in situ hybridization to reveal the expression, functions, and regulatory interactions of the Hox genes in the milkweed bug Oncopeltus fasciatus. Our results reveal many similarities and some interesting differences compared to Drosophila. We find that the gene Antennapedia is required for the identity of all three thoracic segments, while Ultrabithorax, abdominal-A and Abdominal-B cooperate to pattern the abdomen. The three abdominal genes exhibit posterior prevalence like in Drosophila, but apparently via some post-transcriptional mechanism. The functions of the head genes proboscipedia, Deformed, and Sex combs reduced were shown previously, and here we find that the complex temporal expression of pb in the labium is like that of other insects, but its regulatory relationship with Scr is unique. Overall, our data reveal that the evolution of insect Hox genes has included many small changes within general conservation of expression and function, and that the milkweed bug provides a useful model for understanding the roles of Hox genes in a direct-developing insect.

Animals↗
PubMed1999

The trp1- delta FA designer deletion for PCR-based gene functional analysis in Saccharomyces cerevisiae.

PCR-based gene deletion and modification are now common techniques for rapid gene manipulation in the yeast Saccharomyces cerevisiae. The techniques work best when the host strain lacks sequence homology to the PCR-amplified selectable markers. One of the most versatile sets of PCR deletion/modification vectors is the pFA system described by Longtine et al.(1998), which is based on both heterologous (kanMX6 and HIS3MX6) and homologous (TRP1) markers. Here we describe the trp1-DeltaFA designer deletion allele that removes precisely from the genome TRP1 sequences carried in the pFA vectors. The trp1-DeltaFA allele can be introduced easily into TRP1 and most trp1 starting strains, and its use increases the frequency of correct integrants when using the pFA system's TRP1-based constructs. Unlike trp1-Delta1, trp1-DeltaFA does not remove neighbouring GAL3 upstream activating sequences and therefore does not interfere with GAL gene induction.

Aldose-Ketose Isomerases↗
PubMed1985

On ras gene function in yeast.

Saccharomyces cerevisiae contains two RAS genes, RAS1 and RAS2. An insertion mutation in RAS2 (ras2::LEU2) does not affect growth on glucose based media but it does prevent growth on media with pyruvate or other noncarbohydrate carbon sources. This defect is pH sensitive and is most severe at pH 7 and above. The ras2::LEU2 mutation also causes markedly higher levels of glycogen in the derepressed phase of growth after glucose exhaustion. Selection for restoration of growth on pyruvate yields unlinked suppressor mutations. Some of the suppressors also reduce glycogen as well as trehalose (the other reserve carbohydrate in yeast) to levels much lower than those of wild-type strains. These suppressor mutations do not suppress the lethality of ras1 ras2 double mutants. The results indirectly accord with yeast RAS2 governing a G protein activity of adenylate cyclase.

Animals↗
PubMed2003

Nitrogenase genes in PCR and RT-PCR reagents: implications for studies of diversity of functional genes.

Studies of the diversity of microorganisms in the environment have been facilitated by use of PCR and reverse transcription PCR (RT-PCR). Inhibition of the PCR by complex sample matrices and low abundance of some target microorganisms require the use of high-sensitivity amplification procedures, involving a large number of cycles or nested PCR methods. Using these methods, we frequently observed contamination of the amplification reagents, including polymerases, by genomic DNA containing nitrogenase (nifH) and rRNA genes. Contaminating genes were sequenced and found to belong to a variety of rRNA clades, but only three major nifH clades. These sequence types included a few nifH sequences reported in previous studies of the environment. Contamination could be reduced by restriction digestion and ultrafiltration of PCR reagents, but efficiency of amplification was also reduced. Our results suggest that studies relying on large numbers of PCR amplification cycles to assess environmental gene diversity should take precautions to assure that clone libraries generated from amplified PCR products are not the result of contaminated PCR reagents.

Artifacts↗
PubMed2004

A plethora of plant serine/arginine-rich proteins: redundancy or evolution of novel gene functions?

Precursor-mRNA (pre-mRNA) processing is an important step in gene expression and its regulation leads to the expansion of the gene product repertoire. SR (serine-arginine)-rich proteins are key players in intron recognition and spliceosome assembly and significantly contribute to the alternative splicing process. Due to several duplication events, at least 19 SR proteins are present in the Arabidopsis genome, which is almost twice as many as in humans. They fall into seven different subfamilies, three of them homologous with metazoan splicing factors, whereas the other four seem to be specific for plants. The current results show that most of the duplicated genes have different spatiotemporal expression patterns indicating functional diversification. Interestingly, most of the SR protein genes are alternatively spliced and in some cases this process was shown to be under developmental and/or environmental control. This might greatly influence gene expression of target genes as also exemplified by ectopic expression studies of particular SR proteins.

Alternative Splicing↗
PubMed2001

The murine genome contains one functional gene and two pseudogenes coding for the 16 kDa proteolipid subunit of vacuolar H(+)-ATPase.

We cloned the genomic genes encoding the murine 16 kDa subunit (proteolipid, PL16) of vacuolar H(+)-ATPase (V-ATPase) and determined their nucleotide sequences. At least three independent genes were found in the murine genome. One gene consisted of three exons and was largely identical in sequence to that of PL16 cDNA reported previously (Hanada et al., Biochem. Biophys. Res. Commun. 176 (1991) 1062). In the 5'-flanking region of this gene, several possible transcriptional cis-elements were found. TATA and CAAT sequences were not found, which is characteristic for promoters of house-keeping genes. The other two genes identified did not contain introns. One of these genes had an open reading frame that potentially encoded PL16 but contained six amino acid substitutions and a frame-shift mutation that would result in a truncated protein unable to participate in V-ATPase activity. The other gene had the same sequence in the reading frame as that in the cDNA. However, this gene contained a polyA sequence at the same position where polyA is normally added to mRNA. The gene also had 15 bp repetitive sequences near the transcription initiation site and next to the polyA sequence. These observations suggest that this gene may have been generated by the insertion of reverse-transcribed double-stranded cDNA, as is usually observed for pseudogenes. In conclusion, there is a single functional PL16 gene and two pseudogenes in the murine genome. It is unlikely that PL16 isoforms contribute to variation in V-ATPase function.

Amino Acid Sequence↗
PubMed2003

Two different novel cis-acting elements of erd1, a clpA homologous Arabidopsis gene function in induction by dehydration stress and dark-induced senescence.

Many plant genes have been shown to be induced by water stress and function in stress tolerance. The erd1 gene has been shown to be upregulated in response to both water stress and etiolation. Promoter studies using the erd1 promoter region fused to the luciferase (LUC) reporter gene in Arabidopsis thaliana were performed to identify the putative cis elements involved. Results indicated that the cis elements, responsible for gene expression during dehydration and etiolation, are separately located in two discrete portions of the erd1 promoter. Base substitution analysis showed that a 14-bp region from -599 to -586, and a myc recognition motif from -466 to -461 are necessary for the induction of LUC activity in dehydrated plants. On the other hand, base substitution analysis revealed that both an abscisic acid responsive element (ABRE)-like sequence (from -199 to -195) and an ACGT sequence (from -155 to -152) are required for an etiolation-induced increase in LUC activity. LUC activity measurements from etiolated transgenic plants incubated in either water, N6-benzyleadenine (BA), or a 1% sucrose solution found that while BA was able to delay the increase in LUC activity seen in water-treated plants, no increase in LUC activity was seen in plants incubated in sucrose. These results indicate that the erd1 promoter contains two different regulatory systems that are involved in upregulation by dehydration stress and dark-induced senescence.

Adenosine Triphosphatases↗
PubMed2000

Inhibition of Plasmodium falciparum clag9 gene function by antisense RNA.

We have previously shown by targeted gene disruption that the clag9 gene of Plasmodium falciparum is essential for cytoadherence to CD36. Here we report inhibition of the function of clag9 by the use of an antisense RNA vector as an alternative to targeted gene disruption. We transfected an antisense construct of clag9 into the P. falciparum clone 3D7 and when the resulting line was cultured in the presence of pyrimethamine it showed 15-fold lower cytoadherence to C32 melanoma cells than the control. Reversion to wildtype upon removal of the introduced plasmid provides direct evidence that the event responsible for the phenotypic change is not at an unrelated site and this approach provides a valuable new tool in malaria transfection technology.

Animals↗
PubMed2004

Functional gene diversity analysis in BTEX contaminated soils by means of PCR-SSCP DNA fingerprinting: comparative diversity assessment against bacterial isolates and PCR-DNA clone libraries.

Developments in molecular biology based techniques have led to rapid and reliable tools to characterize microbial community structures and to monitor their dynamics under in situ conditions. However, there has been a distinct lack of emphasis on monitoring the functional diversity in the environment. Genes encoding catechol 2,3-dioxygenases (C23O), as key enzymes of various aerobic aromatic degradation pathways, were used as functional targets to assess the catabolic gene diversity in differentially BTEX contaminated environments by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Site specific PCR-SSCP fingerprints were obtained, showing that gene diversity experienced shifts correlated to temporal changes and levels of contamination. PCR-SSCP enabled the recovery of predominant gene polymorphs, and results closely matched with the information retrieved from random sequencing of PCR-DNA clone libraries. A new method for isolating strains capable of growing on BTEX compounds was developed to diminish preselection or enrichment bias and to assess the function of predominant gene polymorphs. C23O abundance in isolates correlated with the levels of BTEX pollution in the soil samples analysed. Isolates harbouring C23O genes, identical to the gene polymorph predominant in all contaminated sites analysed, showed an unexpected benzene but not toluene mineralizing phenotype whereas isolates harbouring a C23O gene variant differing by a single point mutation and observed in highly polluted sites only, were capable, among some other isolates, to mineralize benzene and toluene, indicating a catabolically determined sharing of carbon sources on-site. The PCR-SSCP technique is thus a powerful tool for assessing the diversity of functional genes and the identification of predominant gene polymorphs in environmental samples as a prerequisite to understand the functioning of microbial communities.

DNA Fingerprinting↗
PubMed1986

Assignment of a processed mouse Aprt pseudogene to the same chromosome as the functional gene.

A novel genetic system has been used to demonstrate that a processed adenine phosphoribosyltransferase (Aprt) pseudogene is located on mouse chromosome 8, which is the same chromosome that carries the functional Aprt gene. A restriction fragment length polymorphism associated with the pseudogene was found to segregate concordantly with chromosome 8 in APRT- mutants of a near-diploid cell line that had lost one copy of the chromosome.

Adenine Phosphoribosyltransferase↗
PubMed1999

The Drosophila melanogaster 60A chromosomal division is extremely dense with functional genes: their sequences, genomic organization, and expression.

We cloned and sequenced genomic DNA contigs spanning over 45 kb, surrounding the insertion site of the P-element that is responsible for the developmental defects in the ken and barbie (ken) mutant of Drosophila. This region harbors 10 functional transcription units, in addition to the already well-characterized TGFbeta-60A gene. These include the genes, undefined 1 (UD1), UD2, and UD3, each coding for proteins of unknown function, the ken gene encoding a new Krüppel-like putative transcription factor, the fly homologues of the mammalian mitochondrial trifunctional enzyme (thiolase), and the TAR DNA-binding protein-43 (TBPH), the first nonvertebrate member of the transmembrane 4 superfamily (TM4SF) gene, a new homeodomain gene, and a gene coding for a putative nuclear binding protein (PNBP) that is homologous to maleless, and a Copia-like element. UD3 exists in an intron of the maleless homologue, yet is expressed independent of it. The UD1 and TM4SF genes orient in a tail-to-tail manner with their 3' untranslated region sequences overlapping over 44 nucleotides. Thus the partial overlap and intraintronic organization permitted dense packing of the functional genes within a short segment of the genome.

Acetyl-CoA C-Acetyltransferase↗
PubMed2005

Potato homologs of Arabidopsis thaliana genes functional in defense signaling--identification, genetic mapping, and molecular cloning.

Defense against pests and pathogens is a fundamental process controlled by similar molecular mechanisms in all flowering plants. Using Arabidopsis thaliana as a model, steps of the signal transduction pathways that link pathogen recognition to defense activation have been identified and corresponding genes have been characterized. Defense signaling (DS) genes are functional candidates for controlling natural quantitative variation of resistance to plant pathogens. Nineteen Arabidopsis genes operating in defense signaling cascades were selected. Solanaceae EST (expressed sequence tag) databases were employed to identify the closest homologs of potato (Solanum tuberosum). Sixteen novel DS potato homologs were positioned on the molecular maps. Five DS homologs mapped close to known quantitative resistance loci (QRL) against the oomycete Phytophthora infestans causing late blight and the bacterium Erwinia carotovora subsp. atroseptica causing blackleg of stems and tuber soft rot. The five genes are positional candidates for QRL and are highly sequence related to Arabidopsis genes AtSGT1b, AtPAD4, and AtAOS. Full-length complementary DNA and genomic sequences were obtained for potato genes StSGT1, StPAD4, and StEDS1, the latter being a putative interactor of StPAD4. Our results form the basis for further studies on the contributions of these candidate genes to natural variation of potato disease resistance.

Amino Acid Sequence↗
PubMed2004

Conservation of B-class floral homeotic gene function between maize and Arabidopsis.

The ABC model of flower development, established through studies in eudicot model species, proposes that petal and stamen identity are under the control of B-class genes. Analysis of B- and C-class genes in the grass species rice and maize suggests that the C- and B-class functions are conserved between monocots and eudicots, with B-class genes controlling stamen and lodicule development. We have undertaken a further analysis of the maize B-class genes Silky1, the putative AP3 ortholog, and Zmm16, a putative PI ortholog, in order to compare their function with the Arabidopsis B-class genes. Our results show that maize B-class proteins interact in vitro to bind DNA as an obligate heterodimer, as do Arabidopsis B-class proteins. The maize proteins also interact with the appropriate Arabidopsis B-class partner proteins to bind DNA. Furthermore, we show that maize B-class genes are capable of rescuing the corresponding Arabidopsis B-class mutant phenotypes. This demonstrates B-class activity of the maize gene Zmm16, and provides compelling evidence that B-class gene function is conserved between monocots and eudicots.

Arabidopsis↗
PubMed2001

Atelocollagen-based gene transfer in cells allows high-throughput screening of gene functions.

We have previously demonstrated that Atelocollagen, used clinically for wound healing, is a reliable safe carrier for gene delivery. To obtain phenotypic changes by gene expression of cDNA, we developed an efficient technique for high-throughput gene transfer and expression screening in mammalian cells in microarrays by precoating a microplate with an Atelocollagen complexed with cDNA to which cells are then seeded. The complexes with a nanoparticle form were efficiently transduced into cells without use of any additional transfection reagent, and they allowed for long-term gene expression without apparent chromosomal integration. The complex spotted onto the well of a microplate was stable for a long period and allowed the cells to transduce and express reporter genes in a dose-dependent manner. We also showed that the present method using Atelocollagen-based gene transfer is applicable to gene medicines such as antisense ODNs and adenovirus vectors. These results suggest that Atelocollagen may be appropriate for general use in high-throughput screening of large sets of gene medicines for functional analyses in mammalian cells.

Adenoviridae↗
PubMed1988

An estrogen-responsive element from the 5'-flanking region of the rat prolactin gene functions in MCF-7 but not in HeLa cells.

We have used MCF-7, the human breast cancer cell line, which is estrogen receptor-positive, and the HeLa cell line, which is estrogen receptor-negative, to study the mechanisms by which estrogen induces prolactin gene transcription. A series of plasmids were constructed which direct the expression of the easily assayed bacterial enzyme chloramphenicol acetyltransferase. We have used these recombinants to show that the estrogen-responsive DNA element (ERE) required for the estrogenic regulation of the rat prolactin gene is functional in MCF-7 cells but not in HeLa cells. Specifically, in MCF-7 cells this element enhances the level of gene activity after estrogen treatment both from its own promoter and from a heterologous (simian virus 40) promoter. Results of these studies also show that in HeLa cells the ERE can mediate estrogenic regulation if cotransfected with a plasmid that can synthesize estrogen receptor.

Breast Neoplasms↗