Establishing a method of vector contamination identification in database sequences.

MOTIVATION: The nucleotide sequence databases are invaluable tools both for the private and the academic research communities, from the retrieval of sequences to homology searching. Several issues related to data quality, such as the existence of sequencing artifacts and errors, are facing the databases. We investigated a major source of these errors, i.e. the presence of vector-contaminated sequences. RESULTS: Using a panel of 180 vector polylinker sequences, we found 0.36% or 3029 vector-matching sequences in GenBank Release 95-96, with an average vector-matching length of 72 nucleotides. The number of vector-contaminated sequences has been growing with the database; however, the percent contamination has remained approximately constant at an average of 0.28% from 1982 to 1996. AVAILABILITY: Access to the database of vector polylinker sequences via sequence similarity searching is available at http://seqsim.ncgr.org/vector/ CONTACT: gas@molinfo.com

Two unique 5' untranslated regions in mRNAs encoding human 14-3-3 zeta: differential expression in hemopoietic cells.

In this report, we describe the identification and characterization of a novel 14-3-3 cDNA using the polymerase chain reaction and the screening of a human bone marrow cDNA library. This cDNA encodes the zeta isoform of 14-3-3 and contains a novel 5' untranslated region (UTR) that is G + C rich and only 50% identical to the 5' UTR in the human placental 14-3-3 zeta cDNA, suggesting that 14-3-3 zeta is encoded by at least two mRNAs. Using specific probes to the 5' UTRs of bone marrow and placental 14-3-3 zeta cDNAs, we studied the expression of each transcript in human hemopoietic cells at various stages of differentiation in the myeloid and lymphoid lineages. Differences in the expression of the bone marrow and placental 14-3-3 zeta transcripts were found, the most notable being the markedly decreased expression of both 14-3-3 zeta transcripts in HL-60 myeloid leukemic cells. Western blot analysis of 14-3-3 zeta levels in HL-60 cells revealed correspondingly decreased levels of 14-3-3 zeta protein compared to Jurkat cells. The differences among cell types of relative expression of the two 14-3-3 transcripts may reflect normal regulatory patterns, while the strikingly decreased expression of both types in HL-60 are more likely to be reflective of its multiple genetic abnormalities which contribute to its transformed phenotype.

The Genome Sequence DataBase (GSDB): improving data quality and data access.

In 1997 the primary focus of the Genome Sequence DataBase (GSDB; www. ncgr.org/gsdb ) located at the National Center for Genome Resources was to improve data quality and accessibility. Efforts to increase the quality of data within the database included two major projects; one to identify and remove all vector contamination from sequences in the database and one to create premier sequence sets (including both alignments and discontiguous sequences). Data accessibility was improved during the course of the last year in several ways. First, a graphical database sequence viewer was made available to researchers. Second, an update process was implemented for the web-based query tool, Maestro. Third, a web-based tool, Excerpt, was developed to retrieve selected regions of any sequence in the database. And lastly, a GSDB flatfile that contains annotation unique to GSDB (e.g., sequence analysis and alignment data) was developed. Additionally, the GSDB web site provides a tool for the detection of matrix attachment regions (MARs), which can be used to identify regions of high coding potential. The ultimate goal of this work is to make GSDB a more useful resource for genomic comparison studies and gene level studies by improving data quality and by providing data access capabilities that are consistent with the needs of both types of studies.

The Genome Sequence DataBase version 1.0 (GSDB): from low pass sequences to complete genomes.

The Genome Sequence DataBase (GSDB) has completed its conversion to an improved relational database. The new database, GSDB 1.0, is fully operational and publicly available. Data contributions, including both original sequence submissions and community annotation, are being accomplished through the use of a graphical client-server interface tool, the GSDB Annotator, and via GIO (GSDB Input/Output) files. Data retrieval services are being provided through a new Web Query Tool and direct SQL. All methods of data contribution and data retrieval fully support the new data types that have been incorporated into GSDB, including discontiguous sequences, multiple sequence alignments, and community annotation.

Association of 14-3-3 proteins with centrosomes.

The 14-3-3 proteins are involved in diverse signal transduction pathways and interact physically with a wide variety of proteins. Here, we report the partial sequence analysis of a human spleen 14-3-3 protein, which was identified as a variant form of the epsilon isoform. A peptide antibody generated to the variant 14-3-3 localizes in the centrosome and spindle apparatus of mouse leukemic FDCP cells by immunofluorescence microscopy. Immunoblots of centrosomes isolated by sucrose density gradient centrifugation of cell lysates disclose only the epsilon and gamma isoforms, while total cellular lysates contain the epsilon, gamma, beta and zeta isoforms of 14-3-3. These data suggest that a subset of total cellular 14-3-3 proteins are localized in the centrosomes and spindle apparatus. A differential localization of the centrosomal 14-3-3 was observed in mouse 3T3 cells. Serum-starved (quiescent) cells lack the centrosomal 14-3-3, but upon serum-stimulation of these quiescent cells, the centrosomal 14-3-3 reappears. We propose that a subset of intracellular 14-3-3 proteins are localized in the centrosome and spindle apparatus, and may in fact, link mitogenic signaling, the cell cycle, and perhaps the centrosome duplication cycle as well.

Identification of enzymatically active Ca2+/calmodulin-dependent protein kinase in centrosomes of hemopoietic cells.

In this study, we report the identification of enzymatically active, multifunctional calcium/calmodulin-dependent protein kinase in centrosomes of FDCP1 cells using subcellular fractionation and immunofluorescence techniques. Centrosomes were isolated from detergent lysates of FDCP cells by sucrose density gradient centrifugation and contain tubulin (M(r) = 58 kDa) and centrin (M(r) = 20 kDa) by immunoblotting. Analysis of these fractions with anti-calcium/calmodulin kinase II antibody revealed the presence of the 52 kDa and 56 kDa doublet corresponding to the alpha and the beta/beta' subunits of the enzyme complex. In vitro kinase reactions with isolated centrosomes and in the presence of calcium and calmodulin results in the phosphorylation of several centrosomal proteins.