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Biomedical subjects

L Cooley

Publications and source records attributed to L Cooley.

At least 55 records · Page 3Linked to original sources
PubMed1988

Insertional mutagenesis of the Drosophila genome with single P elements.

A versatile genetic method for identifying and cloning Drosophila melanogaster genes affecting any recognizable phenotype is described. Strains are constructed in which the insertion of a single P transposable element has caused a new mutation, greatly simplifying the genetic and molecular analysis of the affected gene. Mutagenesis is initiated by crossing two strains, each of which contains a specially designed P element. One element (jumpstarter), encoding P element transposase, efficiently mobilizes the second nonautonomous transposon (mutator), whose structure facilitates selection and cloning of new insertion mutations. Random mutator transpositions are captured in individual stocks that no longer contain jumpstarter, where they remain stable. This method was used to construct 1300 single P element insertion stocks which were then screened for recessive mutations. A library of single-element insertion strains will allow the structure and function of Drosophila genes to be readily correlated, and should have many other applications in Drosophila molecular genetics.

Animals↗
PubMed1987

Amplification of the X-linked Drosophila chorion gene cluster requires a region upstream from the s38 chorion gene.

Genomic sequences controlling follicle cell-specific amplification of the X-linked Drosophila chorion gene cluster were mapped by P element-mediated transformation. Several DNA fragments containing the s38 gene and flanking sequences induced tissue-specific amplification, although replication levels were subject to position effects. Deletion analysis identified a 467-bp region upstream from the s38 transcription start site that contained sequences essential in cis for amplification. The essential region shared 32 bp of imperfect sequence homology with a previously identified region necessary for third chromosome chorion gene cluster amplification. This homologous segment contained a repetitive motif consisting of perfect and imperfect AATAC repeats; it was localized near the boundary of the essential domain since most, but not all, the repeats could be deleted without eliminating transposon-induced amplification. The repetitive region was not required for developmentally regulated s38 transcription, therefore our results identified at least one element required for amplification but not for chorion gene transcription. The homologous repetitive sequences within the amplification-essential regions may constitute part of the replication origins used to differentially replicate the two chorion domains during oogenesis.

Animals↗
PubMed1987

Postprandial blush in multiphase bone scanning.

The presence of transient soft-tissue activity in the left side and the lower midportion of the abdomen on the early phases of the multiphase bone scan represents postprandial physiologic hyperemia of the small intestine. The bowel uptake was present in all 33 patients ingesting food between 15 min and 3.5 hr before scanning. In those patients who had not eaten within 4 hr of the study, only 25% demonstrated bowel activity. The observation of bowel uptake is important in differentiating a physiologic phenomenon from pathologic accumulations of activity. Pathology should be ruled out when bowel activity is not located in the usual left flank and lower mid-abdomen, or is present in a fasting individual.

Adolescent↗
PubMed1986

The additional guanylate at the 5' terminus of Escherichia coli tRNAHis is the result of unusual processing by RNase P.

In eucaryotes the 5'-terminal guanylate moiety of mature tRNAHis is added posttranscriptionally. To determine whether the same mechanism occurs in procaryotes, we processed in vitro-derived Escherichia coli tRNAHis precursors to mature tRNA, either in E. coli extracts or by using pure M1-RNA, the catalytic component of RNase P. The results show that the extra guanylate at the 5' end of mature E. coli tRNAHis is encoded in the gene and is found in tRNA as the result of an unusual cleavage by RNase P.

Base Sequence↗
PubMed1985

Processing of precursor tRNAs in Drosophila. Processing of the 3' end involves an endonucleolytic cleavage and occurs after 5' end maturation.

Transfer RNA biosynthesis is a complex process which includes size trimming and nucleotide modification of an initial tRNA precursor. We have examined the temporal order and the nature of tRNA processing events in a Drosophila in vitro transcription/processing system using Drosophila tRNA genes as templates. RNA sequence analysis of processing products indicates that processing at both 5' and 3' ends occurs by endonucleolytic cleavage. The time course of processing of an initial tRNA precursor to mature tRNA reveals that trimming at the 5' end precedes 3' end maturation.

Animals↗
PubMed1985

Structure and transcription of eukaryotic tRNA genes.

The availability of cloned tRNA genes and a variety of eukaryotic in vitro transcription systems allowed rapid progress during the past few years in the characterization of signals in the DNA-controlling gene transcription and in the processing of the precurser RNAs formed. This will be the subject matter discussed in this review.

Animals↗
PubMed1984

Transcriptionally active and inactive gene repeats within the D. melanogaster 5S RNA gene cluster.

Transcription of isolated repeat units of D. melanogaster 5S DNA in a Drosophila KcO cell extract revealed three types of template activities. 5SI DNA encodes the known 5S rRNA of D. melanogaster and has a relatively high transcription efficiency. 5SII DNA is identical to 5SI DNA except for a two-nucleotide deletion at 5S rRNA positions 28 and 29; the efficiency of transcription is approximately 40% that of 5SI DNA and because of the deletion, the primary transcript is two nucleotides shorter. 5SIII DNA does not support in vitro transcription (less than 2% 5SI DNA), but has the same sequence as 5SI DNA except for a single G to A transition at position 86. This is the first reported point-mutation in a 5S RNA gene resulting in loss of transcription function. Of approximately 23 5S rRNA gene copies in a cloned 5S DNA sub-cluster (p12D1) 19 appear to be of the transcriptionally inactive 5SIII DNA type.

Animals↗
PubMed1984

The extent of a eukaryotic tRNA gene. 5'- and 3'-flanking sequence dependence for transcription and stable complex formation.

We have examined the 5'-and 3'-flanking sequence requirements for the "wild type transcription properties" of a Drosophila tRNA Arg gene through the use of transcription assays in cell-free extracts. Thirty-three base pairs of the 5' flank immediately adjacent to the sequence encoding the mature tRNA are necessary for efficient transcription in Drosophila Kc cell extract. Sequences affecting factor binding to form stable transcription complexes extend more than 60 base pairs into the 5' flank, and approximately 35 base pairs into the 3' flank. HeLa cell extract exhibits dependence, albeit reduced, on the same 5'-flanking sequence; it also has 3'-flanking sequence requirements for maximal stable complex formation. This requirement of in vitro transcription for flanking sequence is not dependent on the use of a homologous system, but is dependent on the cellular source of the extract.

Animals↗
PubMed1984

Transcription factor binding is limited by the 5'-flanking regions of a Drosophila tRNAHis gene and a tRNAHis pseudogene.

We determined the sequence of a Drosophila tRNA gene cluster containing a tRNAHis gene and a tRNAHis pseudogene in close proximity on the same DNA strand. The pseudogene contains eight consecutive base pairs different from the region of the bona fide gene which codes for the 3' portion of the anticodon stem of tRNAHis. The tRNAHis gene is transcribed efficiently in Drosophila Kc cell extract, whereas the pseudogene is not. The pseudogene is also a much poorer competitor than the real gene in a stable transcription complex formation assay, even though the sequence alteration in the pseudogene does not affect the sequence or spacing of the putative internal transcription control regions. Recombinant clones were constructed in which the 5'-flanking regions are exchanged. The transcription efficiencies and competitive abilities of the recombinant clones resemble those of the genes from which the 5' flank was derived; for example, the tRNAHis pseudogene with the 5'-flanking sequence of the tRNAHis gene is now efficiently transcribed. Deletion analysis of the pseudogene 5' flank failed to uncover an inhibitory element. Deletion analysis of the real gene showed very high dependence on the presence of the wild-type 5'-flanking sequence for factor binding to the internal control regions and stable complex formation. The 5'-flanking sequence of a Drosophila tRNAArg gene active in the Drosophila Kc cell extract does not restore transcriptional activity or stable complex formation. The tRNAHis gene and pseudogene behave atypically in HeLa cell extract. Both genes compete for HeLa transcription factors, but neither of them is efficiently transcribed. Removal of the 5'-flanking sequences of each gene and replacement with various sequences, including the tRNAArg gene 5' flank, does not allow increased transcription in HeLa cell extract.

Animals↗
PubMed1982

Nonsense suppression in Schizosaccharomyces pombe: the S. pombe Sup3-e tRNASerUGA gene is active in S. cerevisiae.

The gene encoding the efficient UGA suppressor sup3-e of Schizosaccharomyces pombe was isolated by in vivo transformation of Saccharomyces cerevisiae UGA mutants with S. pombe sup3-e DNA. DNA from a clone bank of EcoRI fragments from a S. pombe sup3-e strain in the hybrid yeast vector YRp17 was used to transform the S. cerevisiae multiple auxotroph his4-260 leu2-2 trp1-1 to prototrophy. Transformants were isolated at a low frequency; they lost the ability to grow in minimal medium after passaging in non-selective media. This suggested the presence of the suppressor gene on the non-integrative plasmid. Plasmid DNA, isolated from the transformed S. cerevisiae cells and subsequently amplified in E. coli, transformed S. cerevisiae his4-260 leu2-2 trp1-1 to prototrophy. In this way a 2.4 kb S. pombe DNA fragment carrying the sup3-e gene was isolated. Sequence analysis revealed the presence of two tRNA coding regions separated by a spacer of only seven nucleotides. The sup3-e tRNASerUGA tRNA gene is followed by a sequence coding for the initiator tRNAMet. The transformation results demonstrate that the cloned S. pombe UGA suppressor is active in S. cerevisiae UGA mutant strains.

Ascomycota↗
PubMed1982

Post-transcriptional nucleotide addition is responsible for the formation of the 5' terminus of histidine tRNA.

All sequenced histidine tRNAs have one additional nucleotide at the 5' end when compared to other tRNA species. Sequence analysis of histidine tRNA genes from Drosophila melanogaster and Schizosaccharomyces pombe showed that the terminal guanylate residue of the mature tRNAs is not encoded by the genes. Analysis of the products from in vitro transcription of these genes in extracts from Drosophila Kc cells demonstrated that the 5'-terminal nucleotide present in the mature tRNA is added post-transcriptionally. The addition reaction requires ATP. A portion of the mature tRNAs are then modified at the 5'-terminal pG. Analysis of the RNA species formed during the in vitro maturation of the Drosophila histidine tRNA primary transcript uncovered the following maturation scheme: (i) the primary transcript is processed by RNase P at the 5' end to form an intermediate precursor; (ii) the 3'-flanking sequence is endonucleolytically removed, and a guanylate moiety is added to the 5' end to form mature-sized histidine tRNA; and (iii) a fraction of the 5'-terminal guanylate residues then undergoes modification. In contrast to the capping of eukaryotic mRNA, the guanylate addition to histidine tRNA results in the formation of a (3'-5')-phosphodiester bond. There are no precedents for the post-transcriptional addition of nucleotides (in phosphodiester linkage) to the 5' end of RNA precursors.

Animals↗
PubMed1981

Characterization of initiation factor eIF-3 from wheat germ.

Initiation factor eIF-3 has been isolated from the 120 mM KCl postribosomal supernatant of wheat germ by chromatography on DEAE-cellulose and phosphocellulose. Glycerol gradient centrifugation and polyacrylamide gel electrophoresis under nondenaturing conditions indicate that the eIF-3 prepared in this manner is at least 85% pure. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate shows that the wheat germ eIF-3 contains 11 polypeptides, ranging in molecular weight from about 25,000 to 120,000. Eight of the polypeptides are present in equimolar amounts; two of the polypeptides are present at molar ratios of about 0.5; and one is present at a molar ratio of about 0.3. The highly purified wheat germ eIF-3 does not prevent the association of wheat germ 40S and 60 S ribosomal subunits to a significant extent. Wheat germ eIF-3 does not increase appreciably the ability of eIF-2 to form a ternary complex with Met-tRNAf and GTP. It does, however, enhance the binding of Met-tRNAf to 40 S ribosomal subunits in the presence of eIF-2 and GTP.

Eukaryotic Initiation Factor-3↗
PubMedDate not supplied

Primary malignant rhabdoid tumor of the central nervous system.

Since the initial description of malignant rhabdoid tumor (MRT) of the kidney by Beckwith in 1978, MRTs have been established as a distinct clinicopathologic entity lacking nephrogenic and myogenic differentiation. MRTs are highly aggressive neoplasms with characteristic histopathologic, immunocytochemical, and ultrastructural features. Many reports have appeared documenting primary extrarenal rhabdoid tumors (ERRTs) occurring at diverse sites, including infratentorial and supratentorial compartments of the central nervous system (CNS). The authors report 2 cases of primary CNS-MRT in young male children (6.5 and 7 years of age) and review the literature on CNS-MRTs. Neuroimaging studies showed an inhomogeneous parasagittal mass in the left anterior parietal region involving the motor strip and attached to the lateral aspect of the superior sagittal sinus in one case, and a right parietal parasagittal tumor with a cystic component in the other case. Metastatic workup, including abdominal CT, was negative in both cases. Histologic examination of the resected tumors showed irregular clusters and nests of cells with variable desmoplasia in both cases. Large areas of tumor necrosis and apoptotic tumor cells were present. Prominent eosinophilic cytoplasmic inclusions and eccentric, indented nuclei with conspicuous nucleoli characterized many of the tumor cells. Diffuse strong vimentin reactivity and focal strong reaction for epithelial membrane antigen (EMA) were demonstrated. Cytogenetic analyses reported a normal male karyotype in one case and an abnormal male karyotype with loss of both normal copies of chromosome 22 and gain of one structurally rearranged chromosome 22 in the other case. Ultrastructural examination displayed tumor cells with avoid to indented nuclei, marginated chromatin, and prominent nucleoli. Intercellular junctions were not found. Masses of cytoplasmic intermediate filaments in a characteristic whorled configuration were present. CNS-MRTs are consistently vimentin positive (100%) and usually EMA positive (90%). Glial fibrillary acidic protein, neuron-specific enolase, and S-100 protein are variably expressed. Markers for myogenous differentiation are invariably absent. Ultrastructural characteristics include aggregates of intermediate filaments. Monosomy 22 occurs in some CNS rhabdoid tumors, while most renal rhabdoid tumors are cytogenetically normal with only isolated cases having del(13q), del(11p), del(22)(q11), and unbalanced reciprocal translocation involving chromosomes 8 and 22. The prognosis for CNS rhabdoid tumors is dismal and almost two-thirds of patients are dead of disease shortly after diagnosis; one-third have been reported to be alive with disease, but have been followed for only short periods; and a single patient is reported to be free of disease at 5 years.

Brain Neoplasms↗