TY - JOUR T1 - Analysis of stage-specific gene expression in the bloodstream and the procyclic form of Trypanosoma brucei using a genomic DNA-microarray. JF - Mol Biochem Parasitol Y1 - 2002 A1 - Diehl, Susanne A1 - Diehl, Frank A1 - El-Sayed, Najib M A1 - Clayton, Christine A1 - Hoheisel, Jörg D KW - Animals KW - Blotting, Northern KW - Escherichia coli KW - Gene expression KW - Gene Expression Profiling KW - Genes, Protozoan KW - HUMANS KW - Life Cycle Stages KW - Molecular Sequence Data KW - Oligonucleotide Array Sequence Analysis KW - Polymerase Chain Reaction KW - Transcription, Genetic KW - Trypanosoma brucei brucei AB -

A microarray comprising 21,024 different PCR products spotted on glass slides was constructed for gene expression studies on Trypanosoma brucei. The arrayed fragments were generated from a T. brucei shotgun clone library, which had been prepared from randomly sheared and size-fractionated genomic DNA. For the identification of stage-specific gene activity, total RNA from in vitro cultures of the human, long slender form and the insect, procyclic form of the parasite was labelled and hybridised to the microarray. Approximately 75% of the genomic fragments produced a signal and about 2% exhibited significant differences between the transcript levels in the bloodstream and procyclic forms. A few results were confirmed by Northern blot analysis or reverse-transcription and PCR. Three hundred differentially regulated clones have been selected for sequencing. So far, of 33 clones that showed about 2-fold or more over-expression in bloodstream forms, 15 contained sequences similar to those of VSG expression sites and at least six others appeared non-protein-coding. Of 29 procyclic-specific clones, at least eight appeared not to be protein-coding. A surprisingly large proportion of known regulated genes was already identified in this small sample, and some new ones were found, illustrating the utility of genomic arrays.

VL - 123 CP - 2 ER - TY - JOUR T1 - Trypanosoma cruzi: RNA structure and post-transcriptional control of tubulin gene expression. JF - Exp Parasitol Y1 - 2002 A1 - Bartholomeu, Daniella C A1 - Silva, Rosiane A A1 - Galvão, Lucia M C A1 - el-Sayed, Najib M A A1 - Donelson, John E A1 - Teixeira, Santuza M R KW - Animals KW - Base Sequence KW - Blotting, Northern KW - DNA, Complementary KW - DNA, Protozoan KW - Gene Expression Regulation KW - Half-Life KW - Life Cycle Stages KW - Molecular Sequence Data KW - RNA Processing, Post-Transcriptional KW - RNA, Messenger KW - RNA, Protozoan KW - Transcription, Genetic KW - Transfection KW - Trypanosoma cruzi KW - Tubulin AB -

Changes in tubulin expression are among the biochemical and morphological adaptations that occur during the life cycle of Trypanosomatids. To investigate the mechanism responsible for the differential accumulation of tubulin mRNAs in Trypanosoma cruzi, we determine the sequences of alpha- and beta-tubulin transcripts and analyzed their expression during the life cycle of the parasite. Two beta-tubulin mRNAs of 1.9 and 2.3 kb were found to differ mainly by an additional 369 nucleotides at the end of the 3' untranslated region (UTR). Although their transcription rates are similar in epimastigotes and amastigotes, alpha- and beta-tubulin transcripts are 3- to 6-fold more abundant in epimastigotes than in trypomastigotes and amastigotes. Accordingly, the half-lives of alpha- and beta-tubulin mRNAs are significantly higher in epimastigotes than in amastigotes. Transient transfection experiments indicated that positive regulatory elements occur in the 3' UTR plus downstream intergenic region of the alpha-tubulin gene and that both positive and negative elements occur in the equivalent regions of the beta-tubulin gene.

VL - 102 CP - 3-4 ER - TY - JOUR T1 - Polyadenylylation in copia requires unusually distant upstream sequences. JF - Proc Natl Acad Sci U S A Y1 - 1991 A1 - Kurkulos, M A1 - Weinberg, J M A1 - Pepling, M E A1 - Mount, S M KW - Animals KW - Base Sequence KW - Blotting, Northern KW - DNA Transposable Elements KW - Drosophila melanogaster KW - Eye Color KW - Molecular Sequence Data KW - Oligonucleotides KW - Polymerase Chain Reaction KW - Regulatory Sequences, Nucleic Acid KW - Repetitive Sequences, Nucleic Acid KW - RNA Processing, Post-Transcriptional KW - RNA, Messenger AB -

Retroviruses and related genetic elements generate terminally redundant RNA products by differential polyadenylylation within a long terminal repeat. Expression of the white-apricot (wa) allele of Drosophila melanogaster, which carries an insertion of the 5.1-kilobase retrovirus-like transposable element copia in a small intron, is influenced by signals within copia. By using this indicator, we have isolated a 518-base-pair deletion, 312 base pairs upstream of the copia polyadenylylation site, that is phenotypically like much larger deletions and eliminates RNA species polyadenylylated in copia. This requirement of distant upstream sequences for copia polyadenylylation has implications for the expression of many genetic elements bearing long terminal repeats.

VL - 88 CP - 8 ER - TY - JOUR T1 - Characterization of enhancer-of-white-apricot in Drosophila melanogaster. JF - Genetics Y1 - 1990 A1 - Peng, X B A1 - Mount, S M KW - Alleles KW - Animals KW - Blotting, Northern KW - DNA Transposable Elements KW - Drosophila melanogaster KW - Eye Color KW - Female KW - Heterozygote KW - Homozygote KW - Male KW - Nucleic Acid Hybridization KW - PHENOTYPE KW - Poly A KW - Reproduction KW - RNA KW - RNA, Messenger KW - Transcription, Genetic AB -

The white-apricot (wa) allele differs from the wild-type white gene by the presence of the retrovirus-like transposable element copia within the transcription unit. Most RNAs derived from wa have 3' termini within this insertion, and only small amounts of structurally normal RNA are produced. The activity of wa is reduced in trans by a semidominant mutation in the gene Enhancer-of-white-apricot (E(wa). Flies that are wa and heterozygous for the enhancer have eyes which are much lighter than the orange-yellow of wa alone while E(wa) homozygotes have white eyes. This semidominant effect on pigmentation is correlated with a corresponding decrease in white RNA having wild type structure, and flies homozygous for E(wa) have increased levels of aberrant RNAs. Three reverant alleles of E(wa) generated by reversion of the dominant enhancer phenotype with gamma radiation are noncomplementing recessive lethals, with death occurring during the larval stage. The effects on wa eye pigmentation of varying doses of the original E(wa) allele, the wild type allele, and the revertant alleles suggest that the original E(wa) allele produces a product that interferes with the activity of the wild type gene and that the revertants are null alleles. We propose that the E(wa) gene product influences the activity of the downstream copia long terminal repeat in 3' end formation.

VL - 126 CP - 4 ER - TY - JOUR T1 - Structure and expression of the Drosophila melanogaster gene for the U1 small nuclear ribonucleoprotein particle 70K protein. JF - Mol Cell Biol Y1 - 1990 A1 - Mancebo, R A1 - Lo, P C A1 - Mount, S M KW - Amino Acid Sequence KW - Animals KW - Base Sequence KW - Blotting, Northern KW - Blotting, Southern KW - Cloning, Molecular KW - DNA KW - Drosophila melanogaster KW - Gene expression KW - Gene Library KW - genes KW - HUMANS KW - Molecular Sequence Data KW - Molecular Weight KW - Oligonucleotide Probes KW - Poly A KW - Ribonucleoproteins KW - Ribonucleoproteins, Small Nuclear KW - RNA KW - RNA, Messenger KW - Sequence Homology, Nucleic Acid KW - Xenopus AB -

A genomic clone encoding the Drosophila U1 small nuclear ribonucleoprotein particle 70K protein was isolated by hybridization with a human U1 small nuclear ribonucleoprotein particle 70K protein cDNA. Southern blot and in situ hybridizations showed that this U1 70K gene is unique in the Drosophila genome, residing at cytological position 27D1,2. Polyadenylated transcripts of 1.9 and 3.1 kilobases were observed. While the 1.9-kilobase mRNA is always more abundant, the ratio of these two transcripts is developmentally regulated. Analysis of cDNA and genomic sequences indicated that these two RNAs encode an identical protein with a predicted molecular weight of 52,879. Comparison of the U1 70K proteins predicted from Drosophila, human, and Xenopus cDNAs revealed 68% amino acid identity in the most amino-terminal 214 amino acids, which include a sequence motif common to many proteins which bind RNA. The carboxy-terminal half is less well conserved but is highly charged and contains distinctive arginine-rich regions in all three species. These arginine-rich regions contain stretches of arginine-serine dipeptides like those found in transformer, transformer-2, and suppressor-of-white-apricot proteins, all of which have been identified as regulators of mRNA splicing in Drosophila melanogaster.

VL - 10 CP - 6 ER -