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Control over the onset of DNA synthesis in fission yeast.

The fission yeast Schizosaccharomyces pombe has been used to identify gene functions required for the cell to become committed to the mitotic cell cycle and to initiate the processes leading to chromosome replication in S-phase. Two gene functions cdc2 and cdc10 must be executed for the cell to traverse 'start' and proceed from G1 into S-phase. Before the completion of these two functions the cell is in an uncommitted state and can undergo alternative developmental fates such as conjugation. A third gene, suc1, has also been identified whose product may interact directly with that of cdc2 at 'start'. The molecular functions of the genes involved in the completion of 'start' have been investigated. The cdc2 gene has been shown to be a protein kinase, suggesting that phosphorylation may be involved in the control over the transition from G1 into S-phase. The biochemical functions of the cdc10 and suc1 gene products have not yet been elucidated. A control at 'start' has also been shown to exist in the budding yeast Saccharomyces cerevisiae. Traverse of 'start' requires the execution of the CDC28 gene function. The cdc2 and CDC28 gene products (lower-case letters represent genes of Schizosaccharomyces pombe, and capital letters genes of Saccharomyces cerevisiae) are functionally homologous, suggesting that the processes involved in traverse of 'start' are highly conserved. An analogous control may also exist in the G1 period of mammalian cells, suggesting that the 'start' control step, after which cells become committed to the mitotic cell cycle, may have been conserved through evolution.

Cell Cycle↗

Molecular cloning of linotte in Drosophila: a novel gene that functions in adults during associative learning.

The linotte (lio) gene was identified in a screen for mutations that disrupted 3 hr memory after olfactory associative learning, without affecting the perception of odors or electroshock. The mutagenesis yielded a transposon-tagged gene disruption, which allowed rapid cloning of genomic DNA. The lio transcription unit was identified via rescue of the lio1 learning/memory defect by induced expression of a lio+ transgene in adults. The perception of odors or electroshock remained normal when the lio+ transgene was expressed in these lio1 flies. Learning/memory remained normal when the lio+ transgene was expressed in wild-type (lio+) flies. The lio gene produces only one transcript, the level of expression of which varies throughout development. Sequence analysis indicates that lio encodes a novel protein.

Amino Acid Sequence↗

The multidrug resistance (mdr1) gene product functions as an ATP channel.

The multidrug resistance (mdr1) gene product, P-glycoprotein, is responsible for the ATP-dependent extrusion of a variety of compounds, including chemotherapeutic drugs, from cells. The data presented here show that cells with increased levels of the P-glycoprotein release ATP to the medium in proportion to the concentration of the protein in their plasma membrane. Furthermore, measurements of whole-cell and single-channel currents with patch-clamp electrodes indicate that the P-glycoprotein serves as an ATP-conducting channel in the plasma membrane. These findings suggest an unusual role for the P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Expression of the two maize TATA binding protein genes and function of the encoded TBP proteins by complementation in yeast.

A single gene encodes the TATA binding protein (TBP) in yeasts and animals. Although two TBP-encoding genes (Tbp) previously were isolated from both Arabidopsis and maize, the expression and in vivo function of the encoded plant TBPs were not investigated. Here, we report that the two highly conserved maize Tbp genes are unlinked and reside within larger, ancestrally duplicated segments in the genome. We find quantitative differences in Tbp1 versus Tbp2 transcript accumulation in some maize tissues. These nonidentical expression patterns may indicate differences in the tissue-specific regulation of these genes, which might allow the two encoded maize TBP isoforms to perform nonoverlapping functions in the plant. In addition, we show that the maize TBP products, unlike animal TBPs, are functionally interchangeable with yeast TBP for conferring yeast cell viability. This is a conclusive demonstration of in vivo activity for a nonyeast TBP protein, and these complementation results point to particular amino acids in TBP that are likely to influence species-specific protein interactions.

Amino Acid Sequence↗

Human SP-A protein variants derived from one or both genes stimulate TNF-alpha production in the THP-1 cell line.

In humans, two functional genes of surfactant protein (SP) A, SP-A1 and SP-A2, and several alleles of each functional gene have been characterized. SP-A is a multimeric molecule consisting of six trimers. Each trimer contains two SP-A1 molecules and one SP-A2 molecule. Until now, it has been unclear whether a single SP-A gene product is functional or whether there are functional differences either among alleles or between single-gene SP-A products and SP-A products derived from both genes. We tested the ability of in vitro expressed SP-A variants to stimulate tumor necrosis factor (TNF)-alpha production by THP-1 cells. We observed that 1) single-gene products and products derived from both genes stimulate TNF-alpha production, 2) there are differences among SP-A1 and SP-A2 alleles in their ability to stimulate TNF-alpha production, and 3) the increases in TNF-alpha production are lower after treatment with the SP-A1 alleles than after treatment with the SP-A2 alleles. Furthermore, coexpressed SP-As from SP-A1 and SP-A2 genes have a higher activity compared with SP-As from individual alleles or mixed SP-As from SP-A1 and SP-A2 genes. These data suggest that the SP-A-induced increases in TNF-alpha levels differ among SP-A variants and appear to be affected by SP-A genotype and whether SP-A is derived from one or both genes.

Alleles↗

The Drosophila posterior-group gene nanos functions by repressing hunchback activity.

The development of the body plan in the Drosophila embryo depends on the activity of maternal determinants localized at the anterior and posterior of the egg. These activities define both the polarity of the anterior-posterior (AP) axis and the spatial domains of expression of the zygotic gap genes, which in turn control the subsequent steps in segmentation. The nature and mode of action of one anterior determinant, the bicoid(bcd) gene product, has recently been defined, but the posterior determinants are less well characterized. At least seven maternally acting genes are required for posterior development. Mutations in these maternal posterior-group genes result in embryos lacking all abdominal segments. Cytoplasmic transplantation studies indicate that the maternally encoded product of the nanos(nos) gene may act as an abdominal determinant, whereas the other maternal posterior-group genes appear to be required for the appropriate localization and stabilization of this signal. Here we show that the lack of the nos gene product can be compensated for by eliminating the maternal activity of the gap gene hunchback (hb). Embryos lacking both of these maternally derived gene products are viable and can survive as fertile adults. These results suggest that the nos gene product functions by repressing the activity of the maternal hb products in the posterior of the egg.

Abdomen↗

Two Arabidopsis thaliana genes encode functional pectin methylesterase inhibitors.

We have identified, expressed and characterized two genes from Arabidopsis thaliana (AtPMEI-1 and AtPMEI-2) encoding functional inhibitors of pectin methylesterases. AtPMEI-1 and AtPMEI-2 are cell wall proteins sharing many features with the only pectin methylesterase inhibitor (PMEI) characterized so far from kiwi fruit. Both Arabidopsis proteins interact with and inhibit plant-derived pectin methylesterases (PMEs) but not microbial enzymes. The occurrence of functional PMEIs in Arabidopsis indicates that a mechanism of controlling pectin esterification by inhibition of endogenous PMEs is present in different plant species.

Amino Acid Sequence↗

Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants.

OBJECTIVES: Completion of both the mouse and human genome sequences in the private and public sectors has prompted comparison between the two species at multiple levels. This review summarizes the cytochrome P450 (CYP) gene superfamily. For the first time, we have the ability to compare complete sets of CYP genes from two mammals. Use of the mouse as a model mammal, and as a surrogate for human biology, assumes reasonable similarity between the two. It is therefore of interest to catalog the genetic similarities and differences, and to clarify the limits of extrapolation from mouse to human. METHODS: Data-mining methods have been used to find all the mouse and human CYP sequences; this includes 102 putatively functional genes and 88 pseudogenes in the mouse, and 57 putatively functional genes and 58 pseudogenes in the human. Comparison is made between all these genes, especially the seven main CYP gene clusters. RESULTS AND CONCLUSIONS: The seven CYP clusters are greatly expanded in the mouse with 72 functional genes versus only 27 in the human, while many pseudogenes are present; presumably this phenomenon will be seen in many other gene superfamily clusters. Complete identification of all pseudogene sequences is likely to be clinically important, because some of these highly similar exons can interfere with PCR-based genotyping assays. A naming procedure for each of four categories of CYP pseudogenes is proposed, and we encourage various gene nomenclature committees to consider seriously the adoption and application of this pseudogene nomenclature system.

Alternative Splicing↗

Molecular cloning and cell cycle-specific regulation of a functional human thymidine kinase gene.

A functional thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene has been molecularly cloned from human DNA. The gene was rescued from a genomic library of TK-deficient mouse L cells transformed to the TK+ phenotype with total HeLa cell DNA. Of 14 overlapping clones, only one contained the intact human TK gene. The cloned recombinant bacteriophage carries a 16-kilobase insert derived entirely from human DNA and is capable of transforming LTK- cells to TK+ with an efficiency of 10 TK+ colonies per ng of DNA per 10(6) cells. Restriction endonuclease mapping shows that the functional human TK gene is at least twice as long as that reported for chicken. A 1.6-kilobase Xho I/EcoRI fragment was subcloned and found to hybridize to a human mRNA of 1.5 kilobases. When introduced into LTK- cells, the cloned human TK gene is regulated in the cell cycle-specific manner characteristic of TK+ mammalian cells. That is, TK activity in synchronized cells increases markedly with the onset of DNA synthesis. The signals governing the S-phase induction of TK activity reside within 16 kilobases of human DNA and are correctly interpreted by mouse cells.

Base Sequence↗

Introduction of silencing-inducing transgenes does not affect expression of known transcripts.

While the RNA interference (RNAi) mechanism has only been discovered a decade ago, RNAi is now often used to study gene function by sequence-specific knockdown of gene expression. However, it is still unknown whether introduction of silencing-inducing transgenes alters the transcriptome. To address this question, genome-wide transcriptional changes in silenced and non-silenced backgrounds were monitored through microarray analysis. No significant transcriptional changes were detected when compared to the non-silenced control. This result was confirmed by real-time polymerase chain reaction analysis of genes known to be involved in RNA silencing. In conclusion, introduction of silencing-inducing constructs does not affect expression of known transcripts in other genes than in those homologous to the targeted ones. Consequently, when gene function is studied by RNAi, the transcriptional changes detected will specifically be the result of knockout of the gene of interest, at least for the genes present on the array used in our study.

Arabidopsis↗

Extensive Introgression Failed to Erode Species Boundaries Among Multiple Sympatric Closely Related Species of Roscoea.

How species boundaries are maintained among sympatric closely related species experiencing gene flow is a puzzling question in evolutionary biology. Although introgression is commonly documented, the dynamics and gene function of introgression have rarely been explored to probe why frequent introgression does not necessarily destroy species boundaries in sympatry. In this study, we employ whole-genome resequencing data to examine introgression among five closely related species of Roscoea that coexist in a 'sky island' with seventeen distinct morphological traits. Our findings reveal that introgression has led to the phylogenomic discordance between nuclear and chloroplast genomes among these morphologically distinct species. Additionally, introgression is predominantly asymmetrical in both intensity and gene function, particularly concerning recombination. Notably, the majority of gene functions associated with introgressed loci are unrelated to reproductive processes. Our results suggest that closely related species with incomplete allele assortment can coexist despite theoretical predictions, highlighting the semipermeable nature of species boundaries as reproductive isolation develops. This provides a critical conceptual framework for understanding the interplay between introgression and species persistence. Our finding offers insights into how related sympatric species boundaries can be maintained in the face of frequent asymmetrical gene introgression.

Sympatry↗

Clustering of genes coding for DNA binding proteins in a region of atypical evolution of the human genome.

Comparison of the human and mouse genomes has revealed that significant variations in evolutionary rates exist among genomic regions and that a large part of this variation is interchromosomal. We confirm in this work, using a large collection of introns, that human chromosome 19 is the one that shows the highest divergence with respect to mouse. To search for other differences among chromosomes, we examine the distribution of gene functions in human and mouse chromosomes using the Gene Ontology definitions. We found by correspondence analysis that among the strongest clusterings of gene functions in human chromosomes is a group of genes coding for DNA binding proteins in chromosome 19. Interestingly, chromosome 19 also has a very high GC content, a feature that has been proposed to promote an opening of the chromatin, thereby facilitating binding of proteins to the DNA helix. In the mouse genome, however, a similar aggregation of genes coding for DNA binding proteins and high GC content cannot be found. This suggests that the distribution of genes coding for DNA binding proteins and the variations of the chromatin accessibility to these proteins are different in the human and mouse genomes. It is likely that the overall high synonymous and intron rates in chromosome 19 are a by-product of the high GC content of this chromosome.

Base Composition↗

Influence of long-term treatment of imidapril on mortality, cardiac function, and gene expression in congestive heart failure due to myocardial infarction.

Although it is generally accepted that the efficacy of imidapril, an angiotensin-converting enzyme inhibitor, in congestive heart failure (CHF) is due to improvement of hemodynamic parameters, the significance of its effect on gene expression for sarcolemma (SL) and sarcoplasmic reticulum (SR) proteins has not been fully understood. In this study, we examined the effects of long-term treatment of imidapril on mortality, cardiac function, and gene expression for SL Na+/K+ ATPase and Na+ -Ca2+ exchanger as well as SR Ca2+ pump ATPase, Ca2+ release channel (ryanodine receptor), phospholamban, and calsequestrin in CHF due to myocardial infarction. Heart failure subsequent to myocardial infarction was induced by occluding the left coronary artery in rats, and treatment with imidapril (1 mg.kg(-1).day(-1)) was started orally at the end of 3 weeks after surgery and continued for 37 weeks. The animals were assessed hemodynamically and the heart and lung were examined morphologically. Some hearts were immediately frozen at -70 degrees C for the isolation of RNA as well as SL and SR membranes. The mortality of imidapril-treated animals due to heart failure was 31% whereas that of the untreated heart failure group was 64%. Imidapril treatment improved cardiac performance, attenuated cardiac remodeling, and reduced morphological changes in the heart and lung. The depressed SL Na+/K+ ATPase and increased SL Na+-Ca2+ exchange activities as well as reduced SR Ca2+ pump and SR Ca2+ release activities in the failing hearts were partially prevented by imidapril. Although changes in gene expression for SL Na+/K+ ATPase isoforms as well as Na+-Ca2+ exchanger and SR phospholamban were attenuated by treatments with imidapril, no alterations in mRNA levels for SR Ca2+ pump proteins and Ca2+ release channels were seen in the untreated or treated rats with heart failure. These results suggest that the beneficial effects of imidapril in CHF may be due to improvements in cardiac performance and changes in SL gene expression.

Angiotensin-Converting Enzyme Inhibitors↗

Differential screening of ovarian cDNA libraries detected the expression of the porcine collagenase inhibitor gene in functional corpora lutea.

cDNA libraries were constructed from porcine granulosa cells of antral follicles as well as functional corpus luteum, and clones encoding stage-specific genes have been isolated by differential screening. A clone specific to the functional stage of corpus luteum was found to encode the porcine collagenase inhibitor gene and the stage-specific expression in luteinizing tissue was confirmed by Northern blot analysis. The complete open reading frame of the porcine collagenase inhibitor was deduced from the nucleotide sequence, and the localization of the product was examined by immunohistochemical staining as well in pig ovary; the inhibitor was detected in the intercellular space of luteal cells and in the connective tissue around blood vessels in the functional corpus luteum.

Amino Acid Sequence↗

Construction of hybrid biphenyl (bph) and toluene (tod) genes for functional analysis of aromatic ring dioxygenases.

Multicomponent enzyme complexes of biphenyl (BP) dioxygenase (Dox) encoded by the gene cluster, bphA1A2A3A4 in Pseudomonas pseudoalcaligenes strain KF707 [Taira et al., J. Biol. Chem. 267 (1992) 4844-4858] and toluene Dox encoded by the gene cluster, todC1C2BA in P. putida strain F1 [Zylstra et al., J. Biol. Chem. 264 (1989) 14940-14946], show high homologies (approx. 60%) for the corresponding subunit component in spite of the fact that they have discrete substrate specificities. We constructed hybrid gene clusters by replacing the gene component(s) between the large and small subunits of terminal Dox in the bph and tod gene clusters, and analyzed the function of a novel hybrid aromatic ring Dox. Escherichia coli cells expressing the hybrid gene clusters, todC1::bphA2A3A4, todC1C2::bphA3A4 and bphA1::todC2::bphA3A4, gained the ability to convert benzene-toluene and their derivatives to the dihydrodiols, indicating that the hybrid terminal Dox composed of TodC1::BphA2 and BphA1::TodC2 forms a functionally active multicomponent Dox associated with ferredoxin (Fer) (BphA3) and Fer reductase (BphA4). Moreover, hybrid Dox (composed of TodC1::BphA2A3A4 and TodC1C2::BphA3A4) showed a wide substrate specificity rather similar to that of the wild-type toluene Dox (TodC1C2BA). On the other hand, the hybrid Dox (BphA1::TodC2::BphA3A4) showed oxidative activities for the same compounds, but the rate of oxidation was dependent upon the substrate. These results suggest that (i) the two subunits of terminal Dox are critically involved in the substrate specificity for BP, benzene and their derivatives, and (ii) the electron transport proteins, Fer and Fer reductase, are exchangeable with one another between the BP Dox and toluene Dox complexes.

Base Sequence↗

Integration of embryonic nephrogenic cells carrying a reporter gene into functioning nephrons.

We developed a procedure to introduce and stably express foreign genes into the kidney. The Lac Z reporter gene encoding the bacterial protein beta-galactosidase was introduced by retrovirus-mediated gene transfer into rat nephrogenic mesenchymal cells, which were induced for 24 h with embryonic spinal cord in vitro. The Lac Z-tagged mesenchymal cells were subsequently transplanted underneath the capsule of the neonatal kidney. Two weeks after transplantation, the Lac Z-tagged cells derived from transplants were identified by their beta-galactosidase expression. Well-differentiated Lac Z positive cells were observed in glomerulus and proximal and distal nephron segments. To determine if the tagged mesenchymal cells developed into functional nephrons, fluorescein isothiocyanate-labeled dextran was infused into transplanted animals before death. We observed that fluorescent apical vesicles were colocalized to beta-galactosidase positive proximal tubular cells, indicating that the transplanted mesenchymal cells were integrated into reabsorbing nephrons. These results show the feasibility of introducing foreign genes into epithelia of functioning nephron segments.

Animals↗

Impact of functional genomics and proteomics on radionuclide imaging.

The assessment of gene function following the completion of human genome sequencing may be performed using radionuclide imaging procedures. These procedures are needed for the evaluation of genetically manipulated animals or newly designed biomolecules, which requires a thorough understanding of physiology, biochemistry, and pharmacology. The experimental approaches will involve many new technologies, including in vivo imaging with single photon emission computed tomography and positron emission tomography. Nuclear medicine procedures may be applied for the determination of gene function and regulation using established and new tracers, or using in vivo reporter genes, such as genes encoding enzymes, receptors, antigens, or transporters. Visualization of in vivo reporter gene expression can be performed using radiolabeled substrates, antibodies, or ligands. Combinations of specific promoters and in vivo reporter genes may deliver information about the regulation of the corresponding genes. Furthermore, protein-protein interactions and activation of signal transduction pathways may be visualized noninvasively. The role of radiolabeled antisense molecules for the analysis of messenger ribonucleic acid (RNA) content has to be investigated. However, possible applications are therapeutic intervention using triplex oligonucleotides with therapeutic isotopes, which can be brought near to specific deoxyribonucleic acid sequences to induce deoxyribonucleic acid strand breaks at selected loci. Imaging of labeled siRNA makes sense if these are used for therapeutic purposes to assess the delivery of these new drugs to their target tissue. Pharmacogenomics will identify new surrogate markers for therapy monitoring, which may represent potential new tracers for imaging. Drug distribution studies for new therapeutic biomolecules are needed at least during preclinical stages of drug development. New treatment modalities, such as gene therapy with suicide genes, will need procedures for therapy planning and monitoring. Finally, new biomolecules will be developed by bioengineering methods, which may be used for the isotope-based diagnosis and treatment of disease.

Animals↗

Plant homeobox genes: many functions stem from a common motif.

Homeodomain-containing transcription factors play a variety of important roles in plant, animal, and fungal development. Mutational and evolutionary analysis is beginning to pinpoint specific roles of homeobox genes in plant meristem function. Even though many different plant homeobox genes encode very similar homeodomain sequences, overexpression studies suggest the existence of several levels of regulation that may specify the functions of the different proteins. Recent analysis of the maize Knotted1 gene and its orthologue in barley demonstrates some of these regulatory processes.

Amino Acid Sequence↗