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Biomedical subjects

A Gasch

Publications and source records attributed to A Gasch.

At least 19 recordsLinked to original sources

Rich probabilistic models for gene expression.

Clustering is commonly used for analyzing gene expression data. Despite their successes, clustering methods suffer from a number of limitations. First, these methods reveal similarities that exist over all of the measurements, while obscuring relationships that exist over only a subset of the data. Second, clustering methods cannot readily incorporate additional types of information, such as clinical data or known attributes of genes. To circumvent these shortcomings, we propose the use of a single coherent probabilistic model, that encompasses much of the rich structure in the genomic expression data, while incorporating additional information such as experiment type, putative binding sites, or functional information. We show how this model can be learned from the data, allowing us to discover patterns in the data and dependencies between the gene expression patterns and additional attributes. The learned model reveals context-specific relationships, that exist only over a subset of the experiments in the dataset. We demonstrate the power of our approach on synthetic data and on two real-world gene expression data sets for yeast. For example, we demonstrate a novel functionality that falls naturally out of our framework: predicting the "cluster" of the array resulting from a gene mutation based only on the gene's expression pattern in the context of other mutations.

Algorithms↗

Rapid detection of Listeria monocytogenes by PCR-ELISA.

A rapid detection system specific for Listeria monocytogenes based upon the polymerase chain reaction was developed. The specificity of the primers and the probe annealing to the coding region of the mpl gene proved positive with the DNA from a total of 103 L. monocytogenes strains, while DNA from another 73 Listeria and non-Listeria strains tested negative. To facilitate detection with large numbers of samples, a microtitre plate assay was established with biotinylated probes. Use of a standard DNA prevented false-negative results when used as an internal amplification control in the PCR-ELISA. As the described method required approximately 5-6 h to be completed it may prove useful in the detection of L. monocytogenes in food.

Bacterial Proteins↗

Identification by PCR of receptor-like protein kinases from Arabidopsis flowers.

We have isolated three receptor-like kinase cDNAs from an Arabidopsis flower cDNA library by PCR using degenerate oligonucleotide primers for conserved domains of protein kinases. Cloning and sequencing of the full-length cDNAs, designated RKF1 to 3 (receptor-like kinase in flowers), showed that the putative extracellular domain of the RKF1 protein contains 13 tandem repeats of leucine-rich sequences and those of RKF2 and RKF3 have no significant homology with other plant sequences. RNA blot analysis revealed that the RKF1 mRNA is highly expressed in stamens while RKF2 and RKF3 mRNAs are present at low levels in all organs examined. In situ localization experiments indicated that the RKF1 mRNA is detectable in early flower primordia and during stamen development. In addition, when fused to a GUS reporter gene, the RKF1 promoter directed high GUS expression in pollen grains. Recombinant RKF1, produced in Escherichia coli, was found to have kinase activity with serine/threonine specificity in vitro.

Amino Acid Sequence↗

Expression of three members of the calcium-dependent protein kinase gene family in Arabidopsis thaliana.

Calcium-dependent protein kinases (CDPKs) belong to a unique family of enzymes containing a single polypeptide chain with a kinase domain at the amino terminus and a putative calcium-binding EF hands structure at the carboxyl terminus. From Arabidopsis thaliana, we have cloned three distinct cDNA sequences encoding CDPKs, which were designated as atcdpk6, atcdpk9 and atcdpk19. The full-length cDNA sequences for atcdpk6, atcdpk9 and atcdpk19 encode proteins with a molecular weight of 59343, 55376 and 59947, respectively. Recombinant atCDPK6 and atCDPK9 proteins were fully active as kinases whose activities were induced by Ca2+. Biochemical studies suggested the presence of an autoinhibitory domain in the junction between the kinase domain and the EF hands structure. Serial deletion of the four EF hands of atCDPK6 demonstrated that the integrity of the four EF hands was crucial to the Ca2+ response. All the three atcdpk genes were ubiquitiously expressed in the plant as demonstrated by RNA gel blot experiments. Comparison of the genomic sequences suggested that the three cdpk genes have evolved differently. Using antibodies against atCDPK6 and atCDPK9 for immunohistochemical experiments, CDPKs were found to be expressed in specific cell types in a temporally and developmentally regulated manner.

Amino Acid Sequence↗

The DIMINUTO gene of Arabidopsis is involved in regulating cell elongation.

We have isolated a recessive mutation named diminuto (dim) from T-DNA transformed lines of Arabidopsis thaliana. Under normal growth conditions, the dim mutant has very short hypocotyls, petioles, stems, and roots because of the reduced size of cells along the longitudinal axes of these organs. In addition, dim results in the development of open cotyledons and primary leaves in dark-grown seedlings. The gene for DIM was cloned by T-DNA tagging. DIM encodes a novel protein of 561 amino acids that possesses bipartite sequence domains characteristic of nuclear localization signals. Molecular and physiological studies indicate that the loss-of-function mutant allele does not abolish the response of seedlings to light or phytohormones, although the inhibitory effect of light on hypocotyl elongation is greater in the mutant than in wild type. Moreover, the dim mutation affects the expression of a beta-tubulin gene, TUB1, which is thought to be important for plant cell growth. Our results suggest that the DIM gene product plays a critical role in the general process of plant cell elongation.

Amino Acid Sequence↗

Mapping of a human rRNA gene in the YAC contig surrounding the SMA candidate gene.

Using the yeast artificial chromosome (YAC) 116 flanking the autosomal recessive spinal muscular atrophy (SMA) gene region, we have screened a human fetal brain cDNA library and isolated the cDNA clone 14-3/9 with an insert size of 2.5 kb. The cDNA clone could be identified as part of the human rRNA gene coding for 28S rRNA with a total size of 5025 bp. The human 28S rRNA is involved in the organization of the 60S ribosomal subparticle and is arranged in a 13-kb pre-rRNA transcription unit that occurs in tandem repeat clusters. Multiple copies of the rRNA gene have been mapped by pulsed field blot hybridization in the YAC contig between YAC 66 and YAC 116, which encompasses the SMA candidate gene, and additionally in the distally localized YAC 153.

Adult↗

Evidence that the cis preference of the Tn5 transposase is caused by nonproductive multimerization.

The transposase (Tnp) of the bacterial transposon Tn5 acts 50- to 100-fold more efficiently on elements located cis to the site of its synthesis compared with those located in trans. In an effort to understand the basis for this cis preference, we have screened for Tnp mutants that exhibit increased transposition activity in a trans assay. Two mutations in the carboxyl terminus were isolated repeatedly. The EK345 mutation characterized previously increases Tnp activity eightfold both in cis and in trans. The novel LP372 mutation, however, increases Tnp activity 10-fold specifically in trans. Combining both mutations increases Tnp activity 80-fold. Interestingly, the LP372 mutation maps to a region shown previously to be critical for interaction with Inh, an inhibitor of Tn5 transposition, and results in reduced inhibition activity by both Tnp and Inh. Tnp also inhibits Tn5 transposition in trans, and this has been suggested to occur by the formation of inactive Tnp multimers. Because Inh and (presumably) Tnp inhibit Tn5 transposition by forming defective multimers with Tnp, the inhibition defect of the trans-active LP372 mutant suggests that the cis preference of Tnp may also be attributable to nonproductive Tnp-Tnp multimerization. In addition, we show that increasing the synthesis of EK345/LP372 Tnp, but not wild-type Tnp, leads to very high levels of transposition, presumably because this altered Tnp is defective in the inhibitory activity of the wild type protein.

Base Sequence↗

Fine mapping of human PI 3-kinase associated p85 alpha transcripts in the YAC contig surrounding the spinal muscular atrophy gene.

During a search for transcribed sequences within the gene region for autosomal recessive spinal muscular atrophy (SMA), two cDNA clones were isolated from a human fetal brain and an adult spinal cord cDNA library, respectively, by use of the cosmid LA96B (LAS96). The clones sized 950 bp and 1733 bp detect a 7.7-kb transcript in all tested human tissues. An additional transcript of 6.6 kb is detectable in brain and kidney, and faintly in skeletal muscle and liver. Using comparative human Northern blot analysis, the isolated LA96B cDNA clones could be identified as parts of the 3' untranslated region from the phosphatidylinositol 3 (PI3)-kinase associated p85 alpha transcripts; these were unknown up to now. The 5' end of the gene was mapped to YAC-EFTA:A, whereas the 3' end was localized within the distal overlapping YAC 85 flanking the SMA candidate gene region.

Adult↗

Crystal structure of TFIID TATA-box binding protein.

The structure of a central component of the eukaryotic transcriptional apparatus, a TATA-box binding protein (TBP or TFIID tau) from Arabidopsis thaliana, has been determined by X-ray crystallography at 2.6 A resolution. This highly symmetric alpha/beta structure contains a new DNA-binding fold, resembling a molecular 'saddle' that sits astride the DNA. The DNA-binding surface is a curved, antiparallel beta-sheet. When bound to DNA, the convex surface of the saddle would be presented for interaction with other transcription initiation factors and regulatory proteins.

Amino Acid Sequence↗

Arabidopsis thaliana contains two genes for TFIID.

The general transcription initiation factor TFIID plays a primary part in the activation of eukaryotic genes transcribed by RNA polymerase II. Binding of TFIID to the TATA box initiates the assembly of other general transcription factors as well as RNA polymerase II at the promoter resulting in a preinitiation complex capable of accurate transcription initiation in vitro. Human TFIID has been shown to interact with various regulatory factors. The observation that stimulation of transcription by different trans-acting factors is mediated through distinct TATA elements led to the suggestion that different types of TFIID may exist in yeast, humans and plants. Here we report the cloning and characterization of two distinct TFIID complementary DNA clones from Arabidopsis thaliana. Furthermore, we have found that TFIID from Arabidopsis and other organisms shows homology to helix-loop-helix proteins.

Amino Acid Sequence↗

A 14 bp promoter element directs the testis specificity of the Drosophila beta 2 tubulin gene.

To analyze the regulation of gene expression during male germ cell development, we investigated the testis-specific expression of the Drosophila beta 2 tubulin gene. Germ line transformation experiments with the upstream region of the D.melanogaster beta 2 tubulin gene fused to the Escherichia coli lacZ gene resulted in the correct tissue specific expression of the reporter gene. Furthermore, we showed that the upstream sequences of the beta 2 tubulin gene of the distantly related species D.hydei can drive the expression of the lacZ gene testis specifically in D.melanogaster flies. A detailed deletion analysis showed that 53 bp of upstream and 23 bp (D.melanogaster) or 29 bp (D.hydei) of leader sequences are sufficient to confer tissue specificity. The short promoter regions contain a 14 bp motif at identical positions in both species, which acts as a position-dependent promoter element. In vitro mutagenesis and subsequent germline transformation experiments revealed that this sequence is the only element necessary for the testis-specific transcription of the beta 2 tubulin gene in Drosophila.

Animals↗

Intron and upstream sequences regulate expression of the Drosophila beta 3-tubulin gene in the visceral and somatic musculature, respectively.

The morphogenetic programs involved in the differentiation of internal organs, such as the muscle system, during Drosophila embryogenesis have remained largely obscure. beta 3-tubulin has proved to be a good marker for mesoderm development as this tubulin isotype is detectable soon after mesoderm formation and during the process of mesoderm differentiation. The beta 3-tubulin gene is expressed in the somatic and pharyngeal musculature, the dorsal vessel, and the visceral musculature. To learn more about the programs underlying mesodermal differentiation, we have started to dissect the regulatory elements of the beta 3-tubulin gene by means of P-element-mediated transformation experiments. We show that expression of the beta 3-tubulin gene in the somatic muscles, the pharyngeal muscles, and the dorsal vessel is mediated by far upstream sequences. We also demonstrate that the first intron of the beta 3-tubulin gene bears a tissue-specific enhancer element that is required for expression in the visceral muscles and that also functions efficiently when cloned downstream of an indicator gene. The separability of elements driving beta 3-tubulin expression in the somatic and visceral mesoderm facilitates the investigation of the different programs involved in regulating the early differentiation of this germ layer.

Animals↗

The expression of beta 1 and beta 3 tubulin genes of Drosophila melanogaster is spatially regulated during embryogenesis.

In Drosophila beta tubulins are encoded by a small gene family and the four members of this family are differentially expressed. mRNAs transcribed from two of these genes, namely the beta 1 and beta 3 tubulin genes, are abundant during embryogenesis. While the beta 1 tubulin gene is constitutively expressed during development, beta 3 mRNA is restricted to two distinct phases: mid embryogenesis and metamorphosis. The transcription initiation sites are identical in both these stages and comparison of presumptive promoter regions reveals no extensive homologies between the genes. In situ localization shows beta 1 tubulin mRNA to be maternally expressed in the nurse cells of the egg chambers and evenly distributed during early embryogenesis. In contrast, during later stages of embryogenesis beta 1 tubulin transcripts are predominantly expressed in neural derivatives. The beta 3 tubulin gene expression is also spatially regulated, beta 3 mRNA being restricted to the mesoderm.

Animals↗

Beta 3 tubulin expression characterizes the differentiating mesodermal germ layer during Drosophila embryogenesis.

During embryogenesis, the beta 3 tubulin gene of Drosophila is transcribed predominantly in the mesoderm. We have raised antibodies specific to the C-terminal domain of the beta 3 tubulin and analysed by immunostaining the distribution of this tubulin isotype during Drosophila embryogenesis. The protein is first detectable in the cephalic mesoderm at maximal germband extension. Shortly afterwards, beta 3 tubulin is expressed in single cells at identical positions of the thoracic and abdominal segments. We suggest that these cells represent muscle pioneer cells of Drosophila. During later embryonic development the somatic musclature, visceral musculature, dorsal vessel and macrophages contain beta 3 tubulin. In dorsalizing mutants dorsal, snail and twist, which do not form a ventral furrow during gastrulation, beta 3 expression is greatly reduced but not completely abolished. Our analysis shows that beta 3 tubulin immunostaining characterizes the differentiation of mesodermal derivatives during embryogenesis.

Animals↗

Cloning and identification of the gene coding for the 140-kd subunit of Drosophila RNA polymerase II.

Genomic clones of Drosophila melanogaster were isolated from a lambda library by cross-hybridization with the yeast gene coding for the 150-kd subunit of RNA polymerase II. Clones containing a region of approximately 2.0 kb with strong homology to the yeast gene were shown to code for a 3.9-kb poly(A)-RNA. Part of the coding region was cloned into an expression vector. A fusion protein was obtained which reacted with an antibody directed against RNA polymerase II of Drosophila. Peptide mapping of the fusion protein yielded a number of spots identical with spots derived from the 140-kd subunit of Drosophila RNA polymerase II. Sequence comparison of a segment of the Drosophila and the corresponding yeast clone yielded a high degree of homology at the protein level also, suggesting that we had isolated the gene coding for the 140-kd subunit of RNA polymerase II from Drosophila. In situ hybridization localized the DmRP140 gene at 88 A/B on chromosome 3 while the DmRP215 gene has previously been localized at 10 C on the X chromosome. Analysis of the transcripts (7.0 and 3.9 kb) in female and male flies shows dosage compensation for the transcription of the DmRP215 gene.

Journal Article↗