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It is widely supposed that the tissue specificity of gene expression indicates gene function. Now, an extensive analysis of gene expression in the mouse reveals that quantitative measurement of expression levels in different tissues can contribute powerfully to the prediction of gene function.
Comparative studies suggest that gene duplication, changes in cis-regulatory elements and changes in protein sequence all contribute to the evolution of Hox gene functions, but the evolutionary dynamics of these changes are probably different. It seems likely that gene duplications arise as neutral changes and acquire an adaptive significance later on. By contrast, some changes in regulatory and protein-coding sequences can have immediate consequences in morphological evolution.
Pseudogenes have been defined as non-functional sequences of genomic DNA that are originally derived from functional genes, but exhibit degenerative features such as premature stop codons and frameshifts that prevent their expression. However, there is increasing evidence that pseudogenes are often evolutionarily conserved and may have retained some functional role or acquired new ones. Pseudogenes may exhibit non-functional features as well as functional ones. We investigate, as a model case, the beta-esterase gene cluster of Drosophila melanogaster that includes the Est-6 gene and the psiEst-6 putative pseudogene. We study four samples derived from natural populations of east Africa (Zimbabwe), Europe (Spain), North America (California), and South America (Venezuela). The level of nucleotide diversity is higher in Africa than in the non-African populations. There is twice more nucleotide diversity in psiEst-6 than in Est-6. Linkage disequilibrium within the beta-esterase gene cluster is strong in non-African samples, but much lower in Africa. The population recombination rate is the same for psiEst-6 and Est-6 in Africa, but significantly different in non-African samples. Intragenic gene conversion events are detected within Est-6 and, with much higher incidence, within psiEst-6; intergenic gene conversion events are rare. The extensive intragenic gene conversion within psiEst-6 can be explained by the invasion of retrotransposons that promote a form of homology-dependent gene conversion upon excision. Tests of neutrality with recombination are significant for the beta-esterase gene cluster in the non-African populations but not in Africa. The Est-6 gene sequences exhibit a well-known allozyme dimorphic structure. The sequences of psiEst-6 are also dimorphic in North and South America, but they do not correspond at all (South America) or only imperfectly (North America) to the Est-6 allozyme dimorphism. Sequence dimorphism is less pronounced in the European and African samples. We suggest that demographic history (bottleneck and admixture of genetically differentiated populations) is the major factor shaping the nucleotide pattern in the beta-esterase gene cluster. However, there are some clear indications of positive selection shaping the distribution of nucleotide polymorphism within the cluster. Intergenic epistatic selection may play an important role in the evolution of the beta-esterase gene cluster, preserving psiEst-6 from degenerative destruction and reflecting its functional interaction with Est-6. The Est-6 gene cluster of D. melanogaster represents an example of a functionally interacting complex ('intergene') in which two components (Est-6 and psiEst-6) or more are required to perform the final function.
As the human genome project approaches completion, the challenge for mammalian geneticists is to develop approaches for the systematic determination of mammalian gene function. Mouse mutagenesis will be a key element of studies of gene function. Phenotype-driven approaches using the chemical mutagen ethylnitrosourea (ENU) represent a potentially efficient route for the generation of large numbers of mutant mice that can be screened for novel phenotypes. The advantage of this approach is that, in assessing gene function, no a priori assumptions are made about the genes involved in any pathway. Phenotype-driven mutagenesis is thus an effective method for the identification of novel genes and pathways. We have undertaken a genome-wide, phenotype-driven screen for dominant mutations in the mouse. We generated and screened over 26,000 mice, and recovered some 500 new mouse mutants. Our work, along with the programme reported in the accompanying paper, has led to a substantial increase in the mouse mutant resource and represents a first step towards systematic studies of gene function in mammalian genetics.
MOTIVATION: Assigning functions for unknown genes based on diverse large-scale data is a key task in functional genomics. Previous work on gene function prediction has addressed this problem using independent classifiers for each function. However, such an approach ignores the structure of functional class taxonomies, such as the Gene Ontology (GO). Over a hierarchy of functional classes, a group of independent classifiers where each one predicts gene membership to a particular class can produce a hierarchically inconsistent set of predictions, where for a given gene a specific class may be predicted positive while its inclusive parent class is predicted negative. Taking the hierarchical structure into account resolves such inconsistencies and provides an opportunity for leveraging all classifiers in the hierarchy to achieve higher specificity of predictions. RESULTS: We developed a Bayesian framework for combining multiple classifiers based on the functional taxonomy constraints. Using a hierarchy of support vector machine (SVM) classifiers trained on multiple data types, we combined predictions in our Bayesian framework to obtain the most probable consistent set of predictions. Experiments show that over a 105-node subhierarchy of the GO, our Bayesian framework improves predictions for 93 nodes. As an additional benefit, our method also provides implicit calibration of SVM margin outputs to probabilities. Using this method, we make function predictions for multiple proteins, and experimentally confirm predictions for proteins involved in mitosis. SUPPLEMENTARY INFORMATION: Results for the 105 selected GO classes and predictions for 1059 unknown genes are available at: http://function.princeton.edu/genesite/ CONTACT: ogt@cs.princeton.edu.
The technology for the control of gene function is necessary not only to study development, but also to study pharmacology and to invent new medicines. Morpholino oligo blocks mRNA translation and, as a result, inhibits gene function, mimicking the mutation of the gene. As the method is quite simple and effective, morpholino oligo has been an essential tool in developmental biology. Caged RNA is an innovative technology evolved in Japan. Caged RNA is reduced in translational activity, whereas illumination of caged RNA with ultraviolet light leads to recovery of translational activity. Using caged RNA, we will be able to activate the translation of any gene at any time and in any cell(s) we want.
To investigate T(Brachyury) gene function, a chimeric analysis of midgestation (9.5-11.5 days post coitum) embryos has been performed. Embryonic stem (ES) cell lines homozygous or heterozygous for the T gene have been introduced into wild-type host embryos by blastocyst injection, and the resulting chimeras scored for morphological abnormality and extent of colonization by T/T cells. As observed previously in earlier stage chimeras (Rashbass, P., Cooke, L. A., Herrmann, B. G. and Beddington, R. S. P. (1991) Nature 353, 348-350), 9.5-11.5 dpc T/T<==>+/+ chimeras exhibit many morphological features of intact T/T mutants. In addition, a dramatic bias of T/T cells towards caudal regions (such as tail and allantois) was observed in all chimeras tested. This is likely to result from accumulation of nascent T/T mesoderm cells with time near the primitive streak, possibly because of altered migration or adhesion properties. T/+ cells colonized rostral regions efficiently, but a slight bias towards the distal end of the tail was still evident. No such bias was observed in control chimeras. The presence of T/T cells in the allantois resulted in its failure to form a correct placental connection and thus arrested later development. In contrast, chimeras in which T/T cells were present predominantly in the tail developed normally but exhibited severe tail abnormalities such as foreshortening, branching and haemorrhagic cavities. Moreover, in these embryos, much higher levels of chimerism were present in the distal end of the tail than in younger (9.5 dpc) embryos. Later in gestation, such abnormal tails probably degenerated, giving rise to neonates with absent or severely abnormal tails but no evidence of chimerism. In situ analysis of T expression in the tail reveals that normally T is expressed highly in the tailbud (the growing portion of the tail) during its elongation between 9.5 and 11.5 dpc. Thus, evidence both from chimeras and from T expression in the tail suggest that T may play a role in the correct deployment of cells emerging from the tailbud.
In order to examine gene function in Streptococcus mutans, we have recently initiated an antisense RNA strategy. Toward this end, we have now constructed and evaluated three Escherichia coli-S. mutans shuttle expression vectors with the fruA and scrB promoters from S. mutans, as well as the tetR-controlled tetO promoter from Staphylococcus aureus. Among these, the tetO/tetR system proved to be the most tightly controlled promoter. By using this shuttle plasmid system, modulation of gene function by inducible antisense RNA expression was demonstrated for comC antisense fragments of different sizes as well as for distinct gtfB antisense fragments. It was demonstrated that the size, but not the relative position, of an antisense DNA fragment is important in mediating the antisense phenomenon. Furthermore, by constructing and screening random DNA libraries with the tet expression shuttle system, 78 growth-retarded transformants harboring antisense DNA fragments were also identified. Almost all of them corresponded to homologous essential genes in other bacteria. In addition, a novel essential gene, the coaE gene, encoding dephospho-coenzyme A kinase, which is involved in the final step of coenzyme A catabolism in S. mutans, was identified and characterized. These results suggest that the antisense RNA strategy can be useful for identifying novel essential genes in S. mutans bacteria as well as further characterizing the physiology (including potential virulence factors) of these organisms.
On pre-infection of the host Escherichia coli B with u.v.-inactivated T3 or T7 phage able to express their early genes (like 0.3), B-specific modification of superinfecting, successfully multiplying viruses does not take place. The ocr gene function (gene 0.3) of T3 and T7 not only prevents host-specific DNA restriction but also modification, probably by inhibiting the same late step in the interaction between the restriction enzyme and DNA.
Virus-induced gene silencing (VIGS) is an attractive method for assaying gene function in species that are resistant to conventional genetic approaches. However, VIGS has been shown to be effective in only a few, closely related plant species. Tobacco rattle virus (TRV), a bipartite RNA virus, has a wide host range and so in principle could serve as an efficient vector for VIGS in a diverse array of plant species. Here we show that a vector based on TRV sequences is effective at silencing the endogenous phytoene desaturase (PapsPDS) gene in Papaver somniferum (opium poppy). We show that this vector does not compromise the growth or reproduction of poppy and the plants did not display viral symptoms. The silencing of PapsPDS resulted in a significant reduction in PapsPDS mRNA and a concomitant photobleached phenotype. The ability to rapidly assay gene function in P. somniferum will be valuable in manipulation of the opiate pathway in this pharmaceutically important species. We suggest that our vacuum infiltration method used to deliver TRV-based vectors into poppy is a promising approach for expanding VIGS to diverse angiosperm species in which traditional delivery methods fail to induce VIGS. Furthermore, these studies demonstrate the utility of TRV-VIGS for probing gene function in a basal eudicot species that is phylogenetically distant from model plant species.
Plant MADS-box genes encode transcriptional regulators that are critical for a number of developmental processes. In the angiosperms (the flowering plants), these include the specification of floral organ identities, flowering time and fruit development. It appears that the MADS box gene family has undergone considerable gene duplication and sequence divergence within the angiosperms. Here I discuss the possibility that these events have allowed the recruitment of these genes to new developmental pathways in particular angiosperm lineages. Recent analyses of sequence changes, expression patterns and, in a few cases, gene function are beginning to provide tantalizing evidence for deciphering when and how such genetic diversification has led to particular morphological innovations. In the future, comparative studies of large numbers of species will be required to assess the extent of such variation as well as to fully understand the mechanisms by which evolution of these developmental regulators has played a role in shaping new morphologies.
Recently we established a simple, effective antisense strategy using a double subgenomic Sindbis (dsSIN) virus expression system to study gene function in mosquitoes. In this study, we further elucidate the effects of antisense nucleotide number and duration of viral infection on mosquito gene silencing efficiency by the dsSIN virus expression system. Over 15 days post virus infection, the degree of parasite melanization was progressively reduced by more than 95%, 75% and 55% in the mosquito Armigeres subalbatus transduced with 600, 147 or 36 bases antisense RNA, targeted to the highly conserved copper binding region of the Ar. subalbatus prophenoloxidase I gene (As-pro-POI), respectively. As the duration of viral infection increased from day 3-15, the degree of parasite melanization progressively decreased in all mosquitoes transduced with antisense RNA, irrespective of the lengths of antisense RNA. Progressive loss of parasite melanization function was found to correlate with down regulation of As-pro-PO expression at both the mRNA and protein activity levels, and reductions in virus titres in mosquitoes transduced with antisense RNA. A small pro-PO RNA (c. twenty-five nucleotides) was identified in mosquitoes transduced with antisense RNA. These data suggest that As-pro-POI gene expression is knocked down by degrading the As-pro-POI mRNA through the RNAi pathway. In conclusion, our study demonstrates that even a short antisense RNA (thirty-six bases) can cause silencing of the As-pro-POI gene, and the effects of endogenous gene silencing by dsSIN expression system on mosquito gene functions can be accumulative.
To determine the potential of DNA array technology for assessing functional gene diversity and distribution, a prototype microarray was constructed with genes involved in nitrogen cycling: nitrite reductase (nirS and nirK) genes, ammonia mono-oxygenase (amoA) genes, and methane mono-oxygenase (pmoA) genes from pure cultures and those cloned from marine sediments. In experiments using glass slide microarrays, genes possessing less than 80 to 85% sequence identity were differentiated under hybridization conditions of high stringency (65 degrees C). The detection limit for nirS genes was approximately 1 ng of pure genomic DNA and 25 ng of soil community DNA using our optimized protocol. A linear quantitative relationship (r(2) = 0.89 to 0.94) was observed between signal intensity and target DNA concentration over a range of 1 to 100 ng for genomic DNA (or genomic DNA equivalent) from both pure cultures and mixed communities. However, the quantitative capacity of microarrays for measuring the relative abundance of targeted genes in complex environmental samples is less clear due to divergent target sequences. Sequence divergence and probe length affected hybridization signal intensity within a certain range of sequence identity and size, respectively. This prototype functional gene array did reveal differences in the apparent distribution of nir and amoA and pmoA gene families in sediment and soil samples. Our results indicate that glass-based microarray hybridization has potential as a tool for revealing functional gene composition in natural microbial communities; however, more work is needed to improve sensitivity and quantitation and to understand the associated issue of specificity.
The expression of HSV-specific gene functions by 22 ts mutants of HSV-1 in 15 complementation groups and 8 ts mutants of HSV-2 in 7 complementation groups has been studied at the nonpermissive temperature. Four cistrons of HSV-1 and three cistrons of HSV-2 with defects in viral DNA and DAN polymerase synthesis were identified. DNA-mutants of HSV-1 revealed a greater alteration in HSV polypeptide synthesis and viral assembly than DNA- mutants of HSV-2. Mutants with apparent defects in structural proteins have been identified for both HSV-1 and HSV-2 as demonstrated by their increased themolability. The general organization of the provisional HSV-1 and HSV-2 linkage maps revealed a similarity in the arrangement of functionally related cistrons, with DNA- mutants being located on the left-hand side of each map and mutants with phenotypic properties similar to those of the wild-type virus, on the right-hand side. An early polypeptide of HSV (VP175, MW 175,000) was found to accumulate in cells infected at the nonpermissive temperature withts mutants of HSV-1 in complementation group B. The VP175 polypeptide was isolated from such cells by a combination of SDS-preparative and analytical disc gel electrophoresis. Antiserum prepared to this polypeptide was found to descriminate between HSV-1 and HSV-2 by immunofluorescence. On the other hand, type-specific gene functions of HSV-1 and HSV-2 were not demonstrated through intertypic complementation and recombination tests with heterologous mutant pairs, whereas mutually exchangeable or common gene functions were readily identified. Eight ts mutants of HSV-2 were used to detect functional HSV genes in hamster embryo cells transformed by HSV-2. Normal hamster cells and SV40-transformed hamster cells failed to support the replication of the mutants at the nonpermissive temperature. However, the replication of two mutants, defective in late functions, was significantly enhanced in two independently derived HSV-2-transformed cell lines. Thus functional HSV genetic information was detected in HSV-2-transformed cells through the use of ts mutants. Moreover, it appears that the information present in both cell lines is not only specific but involves late HSV functions.
Previous studies on computational gene functional prediction have not fully exploited the taxonomy structure of Gene Ontology (GO). They just select a few classes from GO into a set, and conduct classwise learning of these classes. The pre-selection of learning classes, often done according to the annotation sizes, limits the prediction breadth and depth. This way of pre-selecting learning classes ignores the taxonomy relations among classes, and so wastes the valuable functional knowledge encoded in the DAG structure of GO. This paper proposes GESTS, a novel gene functional prediction approach based on both gene expression similarity and GO taxonomy similarity, which circumvents the problem of arbitrary learning class pre-selection. GESTS is a semi-supervised approach that reasonably and efficiently incorporates the ontology-formed gene functional knowledge into automated functional analyses of local gene clustering. By integrating both expression similarity and taxonomy similarity into the learning process, GESTS achieves better prediction breadth, depth, and precision than previous studies on the fibroblast serum response dataset and the yeast expression dataset.
Granulocyte colony-stimulating factor is a cytokine which specifically regulates the production of neutrophilic granulocytes. The granulocyte colony-stimulating-factor receptor (GCSFR) is mainly expressed in neutrophils and their precursor cells. In this study, we isolated the chromosomal gene for murine GCSFR and determined its structure. Like the human GCSFR gene homolog, it consists of 17 exons. The exon-intron organization of the murine and human GCSFR-encoding genes are very similar, except that exon 14 and exon 15 in the murine gene are interrupted by a larger intron (greater than 10 kbp) than that found in the human gene (128 bp). This GCSFR-encoding functional gene (Csfgr) was localized to the distal region of murine chromosome 4 by interspecific backcross mapping. A comparison of the 5' flanking sequence of murine and human Csfgr revealed that a sequence of approximately 300 bp upstream from the cap site is highly conserved. Within this region, an 18-nucleotide element conserved in the promoter of the genes for neutrophil-specific enzymes, was found approximately 140 bp upstream from the cap site, suggesting an involvement of this element in the specific expression of GCSFR in neutrophilic granulocytes. In addition to the functional GCSFR-encoding gene, we isolated a pseudogene for GCSFR, which is flanked by a 15-bp direct repeat at the 5' and 3' ends, and lacks all introns, exons 1-3 and exons 7-8 of the functional gene. The processed pseudogene has, in its most 5' region, a sequence of approximately 200 bp that is highly related to the DNA sequence approximately 1.2 kbp upstream of the cap site of the functional gene.
RNA interference (RNAi) has become a well-established technique to study gene function in several species. Our objective was to develop a RNAi approach to study gene function in bovine oocytes. In the first experiment, three different treatments including a 20 min exposure to cytochalasin B, a 6 hr maturation in cycloheximide, and a combination of these two treatments were tested to improve oocyte survival following microinjection. The cycloheximide/cytochalasin B treatment greatly increased (P<0.02) the survival rate of the microinjected oocytes. In the second experiment, we assessed the effect of both cyclin B1 and GFP dsRNA on cyclin B1 mRNA and protein expression. The injection of cyclin B1 dsRNA resulted in a decrease in cyclin B1 mRNA and protein, while the cyclin B2 mRNA remained unaffected. Furthermore, the injection of GFP dsRNA did not interfere with cyclin B1 mRNA or protein nor with the ability of the oocyte to mature properly. In addition, the lack of cyclin B1 in the oocyte led to activation in 10% of the oocytes as evidenced by the presence of a pronucleus. However, the use of an additional 10 hr of maturation in the presence of 6-dimethylaminopurine (6-DMAP) prevented germinal vesicle breakdown and allowed a longer exposure to dsRNA. This procedure increased the percentage of activated oocytes to 33% and is likely to result from an increased length of time for dsRNA processing and for degradation of the cyclin B1 mRNA to occur. In conclusion, RNAi represents a useful technique to study gene function in the bovine oocyte.
The analysis of functional diversity and its dynamics in the environment is essential for understanding the microbial ecology and biogeochemistry of aquatic systems. Here we describe the development and optimization of a DNA microarray method for the detection and quantification of functional genes in the environment and report on their preliminary application to the study of the denitrification gene nirS in the Choptank River-Chesapeake Bay system. Intergenic and intragenic resolution constraints were determined by an oligonucleotide (70-mer) microarray approach. Complete signal separation was achieved when comparing unrelated genes within the nitrogen cycle (amoA, nifH, nirK, and nirS) and detecting different variants of the same gene, nirK, corresponding to organisms with two different physiological modes, ammonia oxidizers and denitrifying halobenzoate degraders. The limits of intragenic resolution were investigated with a microarray containing 64 nirS sequences comprising 14 cultured organisms and 50 clones obtained from the Choptank River in Maryland. The nirS oligonucleotides covered a range of sequence identities from approximately 40 to 100%. The threshold values for specificity were determined to be 87% sequence identity and a target-to-probe perfect match-to-mismatch binding free-energy ratio of 0.56. The lower detection limit was 10 pg of DNA (equivalent to approximately 10(7) copies) per target per microarray. Hybridization patterns on the microarray differed between sediment samples from two stations in the Choptank River, implying important differences in the composition of the denitirifer community along an environmental gradient of salinity, inorganic nitrogen, and dissolved organic carbon. This work establishes a useful set of design constraints (independent of the target gene) for the implementation of functional gene microarrays for environmental applications.