Genetic Expression Analysis Research Articles

Genetic contributions to expression of the baboon cingular remnant

Primitive mammalian molar morphology is characterised in part by a ridge of enamel that encircles the entire base of the molar crown, the cingulum. Many higher primates have reduced the cingulum, but often retain remnant features on the lingual surface of maxillary molars and the labial surface of mandibular molars. Two of these remnants in cercopithecoid primates, the interconulus and interconulid, are morphologically similar though the interconulus is found on maxillary molars and the interconulid is located on mandibular molars. Here we present results from a quantitative genetic analysis of expression of these two traits in a sample of 479 modern savannah baboons from the Southwest Foundation for Biomedical Research (SFBR). We found that both traits are significantly heritable with little variance attributable to other factors, such as sex, age, and molar crown size. Bivariate analyses yielded point estimates for genetic correlations between left and right side expression that are either equal to or not significantly different from 1.0; meaning that 100% of their additive genetic variance is due to the effects of the same gene or suite of genes. By contrast, our estimates of the genetic correlations between maxillary and mandibular expression of this trait range from 0.52 to 0.72, suggesting that 28–52% of the additive genetic variance in the interconulus and interconulid is due to the effects of shared genes. These results demonstrate that intra-arch expression is characterised by complete pleiotropy whereas inter-arch expression is caused by incomplete pleiotropy. These results are relevant to dental developmental studies as well as paleontological analyses of the evolution of the primate dentition.

Culturing Mouse Peritoneal Mesothelial Cells

Obtaining normal cells has become increasingly important for use in comparative genetic analytical techniques to examine alterations in gene expression during transformation and progression into malignancy. Normal mesothelial cells are not currently available in cell banks and are essential for comparison of genetic expression analysis in current mouse mesothelioma models. The purpose of this investigation was to extract normal mouse peritoneal mesothelial cells using minimal culture techniques to obtain sufficient cells for gene expression analysis. Mesothelial cells were collected from the mouse peritoneal cavity in vivo with minimal contamination of lymphocytes and macrophages. The cells were cultured for approximately eight days until they were just confluent and retained normal mesothelial phenotype and cytokeratin immunoperoxidase staining.

A cuticle collagen encoded by the lon-3 gene may be a target of TGF-beta signaling in determining Caenorhabditis elegans body shape.

The signaling pathway initiated by the TGF-beta family member DBL-1 in Caenorhabditis elegans controls body shape in a dose-dependent manner. Loss-of-function (lf) mutations in the dbl-1 gene cause a short, small body (Sma phenotype), whereas overexpression of dbl-1 causes a long body (Lon phenotype). To understand the cellular mechanisms underlying these phenotypes, we have isolated suppressors of the Sma phenotype resulting from a dbl-1(lf) mutation. Two of these suppressors are mutations in the lon-3 gene, of which four additional alleles are known. We show that lon-3 encodes a collagen that is a component of the C. elegans cuticle. Genetic and reporter-gene expression analyses suggest that lon-3 is involved in determination of body shape and is post-transcriptionally regulated by the dbl-1 pathway. These results support the possibility that TGF-beta signaling controls C. elegans body shape by regulating cuticle composition.

Analysis of acute myelogenous leukemia: preparation of samples for genomic and proteomic analyses.

During the last decade, several large clinical studies have demonstrated that analysis of chromosomal abnormalities is an essential basis for therapeutic decisions in patients with acute myelogenous leukemia (AML), and cytogenetic studies should now be regarded as mandatory both for routine treatment and as a part of clinical investigations in AML. However, new techniques for detailed genetic characterization and analysis of gene expression as well as protein modulation will become important in the further classification of AML subsets and the development of risk-adapted therapeutic strategies. In this context, we emphasize the importance of population-based clinical studies as a basis for future therapeutic guidelines. Such studies will then require the inclusion of patients at small clinical centers without specialized hematological research laboratories. To document a high and uniform quality of the laboratory investigations, it will be necessary to collect material for later analysis in selected laboratories. In this article, we describe current methods for collection of biological samples that can be used for later preparation of DNA, RNA, and proteins. With the use of gradient-separated AML cells, it should be possible to establish the necessary techniques for collection and handling of biological samples even at smaller centers, and complete collections from all included patients should then be possible even in population-based clinical studies.

The genetic architecture of quantitative traits.

Phenotypic variation for quantitative traits results from the segregation of alleles at multiple quantitative trait loci (QTL) with effects that are sensitive to the genetic, sexual, and external environments. Major challenges for biology in the post-genome era are to map the molecular polymorphisms responsible for variation in medically, agriculturally, and evolutionarily important complex traits; and to determine their gene frequencies and their homozygous, heterozygous, epistatic, and pleiotropic effects in multiple environments. The ease with which QTL can be mapped to genomic intervals bounded by molecular markers belies the difficulty in matching the QTL to a genetic locus. The latter requires high-resolution recombination or linkage disequilibrium mapping to nominate putative candidate genes, followed by genetic and/or functional complementation and gene expression analyses. Complete genome sequences and improved technologies for polymorphism detection will greatly advance the genetic dissection of quantitative traits in model organisms, which will open avenues for exploration of homologous QTL in related taxa.

The structural and functional organization of the yeast mediator complex.

The Mediator complex of Saccharomyces cerevisiae is required for diverse aspects of transcription by RNA polymerase II (pol II). Mediator is composed of two functionally distinct subcomplexes, Rgr1 and Srb4. To identify the structures and functions of each subcomplex, we expressed recombinant proteins for each subunit and assayed their interactions with each other and with basal transcription proteins. The Rgr1 subcomplex is composed of the Gal11 module, which binds activators, and the Med9/10 module. The Med9/10 module is required for both transcriptional activation and repression, and these activities appear to be carried out by two submodules. Proteins in the Med9 submodule interact physically and genetically with Srb10/11, suggesting that the Med9 submodule mediates the repression of pol II. Purified recombinant Srb4 subcomplex stimulated basal transcription of pol II but had little effect on activated transcription and phosphorylation of the C-terminal domain of the Rpb1 subunit of pol II. Both subcomplexes of Mediator interacted with a distinct set of basal transcription factors and pol II. The modular organization of Mediator and the associated functions suggest that the Mediator complex may recruit and/or stabilize the preinitiation complex through several points of contact with transcriptional regulators and basal transcription factors.

Sequence analysis of libraries from individual human blastocysts.

It has recently become possible to construct cDNA libraries from individual human blastocysts to investigate the expression of embryonic genes in human preimplantation development. We have previously reported the expression of beta-actin, CD-59, and homeobox OCT-3 and identified almost-complete homology of sequences to human histone 3.1 and human ribosome protein S25. In the present paper, our further sequencing analysis of cDNA libraries from single human blastocysts is described. cDNA libraries were constructed from 13 blastocysts. Sequence analysis was performed in 120 clones from one of these cDNA libraries with fragments of 50 to 1000 bp. Their sequence identity was analyzed using the expressed sequence tag (EST) database. The presence of two housekeeping genes, hexokinase I and serine/threonine phosphorylase, and four other ESTs was demonstrated, the identity of which, with particular gene expression in preimplantation development, has not yet been established. The data demonstrate the usefulness of constructing cDNA libraries from individual human blastocysts and their values in the analysis of genetic expression in human preimplantation development.

Yeast microarrays for genome wide parallel genetic and gene expression analysis.

We have developed high-density DNA microarrays of yeast ORFs. These microarrays can monitor hybridization to ORFs for applications such as quantitative differential gene expression analysis and screening for sequence polymorphisms. Automated scripts retrieved sequence information from public databases to locate predicted ORFs and select appropriate primers for amplification. The primers were used to amplify yeast ORFs in 96-well plates, and the resulting products were arrayed using an automated micro arraying device. Arrays containing up to 2,479 yeast ORFs were printed on a single slide. The hybridization of fluorescently labeled samples to the array were detected and quantitated with a laser confocal scanning microscope. Applications of the microarrays are shown for genetic and gene expression analysis at the whole genome level.

In vivo DNA expression of functional brome mosaic virus RNA replicons in Saccharomyces cerevisiae.

To facilitate manipulation of brome mosaic virus (BMV) RNA replicons in Saccharomyces cerevisiae and for yeast genetic analysis of BMV RNA replication, gene expression, and host interactions, we constructed DNA plasmids from which BMV RNA3 and RNA3 derivatives can be transcribed in vivo from the galactose-inducible yeast GAL1 promoter and terminated by a self-cleaving ribozyme at or near their natural 3' ends. In galactose-induced yeast harboring such plasmids, expression of BMV RNA replication proteins 1a and 2a led to synthesis of negative-strand RNA3, amplification of positive-strand RNA3 to levels over 45-fold higher than those of DNA-derived RNA3 transcripts, and synthesis of the RNA3-encoded subgenomic mRNA for coat protein. Although the GAL1 promoter initiated transcription from multiple sites, 1a and 2a selectively amplified RNA3 with the authentic viral 5' end. As expected, reporter genes substituted for the 3'-proximal coat protein gene could not be translated directly from DNA-derived RNA3 transcripts, so their expression depended on 1a- and 2a-directed subgenomic mRNA synthesis. In yeast in which DNA transcription of B3CAT, an RNA3 derivative with the chloramphenicol acetyltransferase (CAT) gene replacing the coat gene, was induced, CAT activity remained near background levels in the absence of 1a and 2a but increased over 500,000-fold when 1a and 2a were expressed. Similarly, a plasmid encoding B3URA3, an RNA3 derivative with the yeast URA3 gene replacing the coat gene, conferred uracil-independent growth to ura3- yeast only after 1a and 2a expression and galactose induction. Once its 1a- and 2a-dependent replication was initiated, B3URA3 was maintained in dividing yeast as a free RNA replicon, even after repression of the GAL1 promoter or the loss of the B3URA3 cDNA plasmid. These findings should be useful for many experimental purposes.

Genetic analysis of renin gene expression in the central nervous system of spontaneously hypertensive rats

Renin expression in the central nervous system in spontaneously hypertensive rats (SHR) has been reported to be higher than that in age-matched Wistar–Kyoto (WKY) rats. In the present study, we examined the mechanisms of enhancement of renin mRNA concentration in the brainstem of SHR using F 2 hybrid rats derived from cross-breeding SHR with WKY. The SHR allele of the renin gene was significantly associated with higher brain renin mRNA concentration in both 10- and 24-week-old F 2 populations. However, neither the genotype of the renin gene nor the brain renin mRNA concentration was associated with blood pressure or urinary volume. Thus, the higher renin mRNA concentration in the brainstem of SHR was indicated to be genetically determined by the genotype of the renin gene. Further study is required to clarify the pathophysiological meaning of this increased renin expression in the brain of SHR.

Biology of Developmental Transcription Control. Conference proceedings. Irvine, California, October 26-28, 1995.

Biology of Developmental Transcription Control. Conference proceedings. Irvine, California, October 26-28, 1995.

Genetic Analysis of Renin Gene Expression in Rat Adrenal Gland

We examined the mechanism of the increased renin mRNA concentration in the adrenal glands of spontaneously hypertensive rats (SHR). In 52 female F2 rats (25 to 27 weeks of age) derived from SHR and Wistar-Kyoto rats, we determined blood pressure, renin mRNA concentration in the adrenal gland, plasma renin activity, plasma aldosterone concentration, and genotype of the renin gene. Eighteen of the F2 rats were fed a high salt (8%) diet for 14 days. The renin mRNA concentration in the adrenal glands showed a significant correlation with the genotype of the renin gene in the normal salt diet group (P <.0001), whereas this relationship was not observed in the high salt group. Multivariate analysis revealed that the plasma aldosterone concentration in the normal diet group was significantly explained (P=.0004, R2=.454) by plasma renin activity (P=.0005), the renin mRNA concentration in the adrenal gland (P=.0496), and the genotype of the renin gene (P=.0236). The SHR allele of the renin gene was associated with a lower aldosterone concentration. On the other hand, in the high salt diet group, only the genotype of the renin gene showed a significant relationship with plasma aldosterone concentration (P=.0237). Again, the SHR allele of the renin gene was associated with a lower aldosterone concentration. We can conclude that the higher renin mRNA concentration in the SHR adrenal glands is governed by the SHR allele of the renin gene or renin gene locus. The renin mRNA concentration in the adrenal gland exerts a minor influence on aldosterone synthesis. Paradoxically, the SHR allele of the renin gene or renin gene locus confers a lower rate of aldosterone synthesis at 25 to 27 weeks of age, the mechanism of which remains to be determined.

The Plastid-encoded ccsA Gene Is Required for Heme Attachment to Chloroplast c-type Cytochromes

A chloroplast gene, ycf5, which displays limited sequence identity to bacterial genes (ccl1/cycK) required for the biogenesis of c-type cytochromes, was tested for its function in chloroplast cytochrome biogenesis in Chlamydomonas reinhardtii. Targeted inactivation of the ycf5 gene results in a non-photosynthetic phenotype attributable to the absence of c-type cytochromes. The cloned ycf5 gene also complements the phototrophic growth deficiency in strain B6 of C. reinhardtii. B6 is unable to synthesize functional forms of cytochromes f and c6 owing to a chloroplast genome mutation that prevents heme attachment. The selected (phototrophic growth) as well as the unselected (holocytochrome c6 accumulation) phenotypes were restored in complemented strains. The complementing gene, renamed ccsA (for c-type cytochrome synthesis), is expressed in wild-type and B6 cells but is non-functional in B6 owing to an early frameshift mutation. Antibodies raised against the ccsA gene product recognize a 29-kDa protein in C. reinhardtii. The 29-kDa protein is absent in strain B6 but is restored in a spontaneous revertant (B6R) isolated from a culture of B6. Sequence analysis of the ccsA gene in strain B6R indicates that it is a true revertant. We conclude that the ccsA gene is expressed and that it encodes a protein required for heme attachment to c-type cytochromes.

Genetic mapping and expression analysis of the murine DNA ligase I gene.

We mapped the murine DNA ligase I gene (Lig1) in the mouse genome by using a mapping panel from an interspecific cross. Lig1 mapped to a centromeric part of chromosome 7, a region homologous to human chromosome 19q, where the human homologue LIG1 was localized. In addition, Lig1 expression was analyzed during the course of mouse liver-cell regeneration induced by partial hepatectomy, necrogenic doses of carbon tetrachloride, or the mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene. The results demonstrate that Lig1 is expressed in the liver during active cell proliferation.

Neurons Producing Specific Neuropeptides in the Central Nervous System of Normal and Pupariation-Delayed Drosophila

Antibodies generated against bombyxin and prothoracicotropic hormone (PTTH) of Bombyx mori and allatotropin, allatostatin, and diuretic hormone (DH) of Manduca sexta react with distinct sets of cells in the central nervous system of Drosophila larvae, pupae, and adults. Brain neurons immunoreactive with antibodies to bombyxin, PTTH, and DH are in strikingly similar positions to their lepidopteran counterparts, indicating that at least some Drosophila neuroendocrine cells are homologous to those of lepidopterans. Allatotropin and allatostatin-immunopositive neurons of Drosophila differ from those of lepidopterans, but many of them are identical with neurons that express the FMRFamide gene. Antibodies to bombyxin, PTTH, allatostatin, and DH also stain axons and axon terminals in the neurohemal part of the ring gland, and all tested antibodies except that against bombyxin show positive reaction in the neurohemal area of the ventral ganglion. Although immunoreactivity with all antibodies is variable during development, the highest levels of staining are found at developmental stages when the neuropeptides would be expected to be functioning. A genetic analysis of neuropeptide expression and function has been initiated by analyzing immunoreactivity in mutants that have prolonged larval life associated with imaginal disc overgrowth. Two of these mutants, dlg and dco, show abnormally strong immunoreactivity for allatotropin during the extended larval period and the former also show increased staining with the PTTH antibody. The reduced ecdysteroid titer and delayed or blocked metamorphosis in the mutants may be a result of altered neuropeptide production, which is probably secondary to the imaginal disc overgrowth.

Genetic analysis of maintenance and expression of L and M double-stranded RNAs from yeast killer virus K28.

The killer phenotype expressed by Saccharomyces cerevisiae strain 28 differs from that of the more extensively studied K1 and K2 killers with respect to immunity, mode of toxin action and cell wall primary toxin receptor. We previously demonstrated that the M28 and L28 dsRNAs found in strain 28 are present in virus-like particles (VLPs) and that transfection with these VLPs is sufficient to confer the complete K28 phenotype on a dsRNA-free recipient cell. We also demonstrated that L28, like the L-A-H species in K1 killers, has [HOK] activity required for maintenance of M1-dsRNA, and predicted that M28 would share with M1 dependence on L-A for replication. We now confirm this prediction by genetic and biochemical analysis of the effects of representative mak, ski and mkt mutations on M28 maintenance, demonstrating that M28 replication resembles M1 in all respects. We also show that L28 is an L-A-H species lacking [B] activity, and that M28 excludes both M1 and M2 from the same cytoplasm. Stable coexpression of K28 phenotype from M28 and of K1 phenotype from an M1-cDNA clone was demonstrated. Exclusion, therefore, acts at the level of dsRNA replication, presumably reflecting competition for the L-A-H encoded capsid and cap-pol fusion protein, rather than reflecting incompatibility of toxin or immunity expression. Finally, we show that expression of active K28 toxin, but not of K28 immunity, requires the Kex2 endoprotease.

Physical and molecular genetic analysis of Qa-2 antigen expression: Multiple factors controlling cell surface levels

We have examined the surface expression of Qa-2 on lymphocyte subpopulations and on splenocytes from inbred mouse strains by a radioactive binding assay using purified anti-Qa-2 antibodies and antibody fragments (Fab). Quantitative measurements by Scatchard analysis revealed that spleen cells from Qa-2 high mice express (4–5) × 10 4 Qa-2 molecules/cell, whereas T lymphocytes have as high as (7–8) × 10 4 molecules/cell. In addition, it was determined that B lymphocytes express (5–6) × 10 3 molecules on their cell surface. The Qa-2 levels on anti-CD3-activated T cells is 1.0 × 10 5 molecules/cell. Previous experiments have shown that the quantity of Qa-2 varies in a strain-specific fashion and may be classified into three groups: Qa-2 high, Qa-2 medium and Qa-2 low. Our results indicated that Qa-2 high strains express (4–5) × 10 4 Qa-2 molecules/cell, Qa-2 medium strains (B6-K2, B10.A, A/J, BALB/c and DBA/2) express (1–1.7) × 10 4 molecules/cell, and Qa-2 low strains (SWR/J and DBA/I) express no more than 6 × 10 3 molecules/cell. Detection of Q7 or Q9 mRNA by the polymerase chain reaction revealed that Qa-2 high strains express two functional Qa-2 enconding genes, Q7 and Q9, whereas Qa-2 mediumand Qa-2 low strains express either Q7 or Q9. These results strongly suggest that Qa-2 gene dosage contributes in part to the variation of Qa-2 levels on the cell surface.

Construction and characterization of a Mycobacterium-Escherichia coli shuttle vector

We have constructed a set of novel Mycobacterium-Escherichia coli shuttle vectors using either a kanamycin- or hygromycin-resistance gene and the replication region from a Corynebacterium plasmid. Important features of these new vectors (pEP2 and pEP3) are that they are small, contain multiple cloning sites, and replicate to high copy number in various Mycobacterium species and E. coli. These vectors are unusual in that plasmid replication in gram-negative and gram-positive bacteria appears to be controlled from a single region. These plasmids will be useful for the genetic analysis of Mycobacterium and gene expression in this genus, particularly Mycobacterium bovis BCG.

Genetic analysis of CR1 expression on erythrocytes of patients with systemic lupus erythematosus

The role of genetic factors in decreased expression of CR1 in patients with systemic lupus erythematosus (SLE) was investigated by assessing the frequency of genotypes determining the numbers of CR1 on erythrocytes obtained from 52 patients with SLE and from 84 normal individuals. The expression of CR1 was quantitated using flow cytometry. Genotypes were determined by analyzing the CR1 gene restriction fragment length polymorphism using the CR1.1 complementary DNA probe and the Hind III restriction enzyme. In normal subjects, the distribution of the HH, HL, and LL genotypes fit the Hardy-Weinberg law. The frequency for the H allele did not differ between the 2 groups. No individual homozygous for the LL genotypes was found among the SLE patient population. Taken together, data from this and previous studies indicate an under-representation of the LL homozygous genotype in patients with SLE. SLE patients expressed decreased numbers of CR1 per erythrocyte within each genotype compared with these numbers in normal subjects. The results suggest that defective expression of CR1 in SLE patients is acquired and that the presence of the L allele is not linked to a genetic susceptibility for SLE.

Genetic mapping and analysis of somatostatin expression in Snell dwarf mice

Mice homozygous for the gene dwarf ( dw) have elevated levels of somatostatin (SS) in extra-hypothalamic brain regions. By in situ hybridization, increased levels of SS mRNA were observed in regions shown previously to contain higher levels of the SS peptide. Thus, the rate of transcription and/or the stability of SS mRNA are affected by the dw mutation. Since both dw and the gene encoding SS, Smst, are located on mouse chromosome 16, two backcrosses segregating dw and Smst were used to determine whether dw is an allele of Smst. In one backcross, an inbred strain derived from the subspecies Mus musculus molossinus was used to provide a high degree of DNA sequence polymorphism. The gene order and map distances determined on this backcross were: (centromere) — Prm-1 — 7 — Igl-1 — 3 — Smst — 29 — dw — 15 — Sod-1 — 4 — Ets-2, demonstrating clearly that Smst and dw are distinct genes. Additional evidence against a primary role for SS excess in the pathogenesis of dw/dw mice was obtained by injecting normal newborn mice with a potent SS analog (cyclo II). In contrast to the pattern of cell loss observed in the dwarf anterior pituitary, the pituitaries of injected mice were indistinguishable from normal controls, further suggesting that the Smst locus is not the primary site of dw gene action.