Possible alternative splicing of the rat SA gene.
Explore the source record for details and available documents.
Alternative Splicing↗
Biomedical subjects
L Gu
Publications and source records attributed to L Gu.
Acetylation of lysine 40 in alpha-tubulin is not essential in Tetrahymena thermophila.
In Tetrahymena, at least 17 distinct microtubule structures are assembled from a single primary sequence type of alpha- and beta-tubulin heterodimer, precluding distinctions among microtubular systems based on tubulin primary sequence isotypes. Tetrahymena tubulins also are modified by several types of posttranslational reactions including acetylation of alpha-tubulin at lysine 40, a modification found in most eukaryotes. In Tetrahymena, axonemal alpha-tubulin and numerous other microtubules are acetylated. We completely replaced the single type of alpha-tubulin gene in the macronucleus with a version encoding arginine instead of lysine 40 and therefore cannot be acetylated at this position. No acetylated tubulin was detectable in these transformants using a monoclonal antibody specific for acetylated lysine 40. Surprisingly, mutants lacking detectable acetylated tubulin are indistinguishable from wild-type cells. Thus, acetylation of alpha-tubulin at lysine 40 is non-essential in Tetrahymena. In addition, isoelectric focusing gel analysis of axonemal tubulin from cells unable to acetylate alpha-tubulin leads us to conclude that: (a) most or all ciliary alpha-tubulin is acetylated, (b) other lysines cannot be acetylated to compensate for loss of acetylation at lysine 40, and (c) acetylated alpha-tubulin molecules in wild-type cells contain one or more additional charge-altering modifications.
Acetylation↗
Generation and characterization of monoclonal antibodies specific for members of the mammalian 70-kDa heat shock protein family.
The 70-kDa heat shock proteins (hsp70) are a highly conserved, abundant, and ubiquitous family of proteins expressed by all organisms from bacteria to humans. It is well established that hsp70 family members function as molecular chaperones and aid in the intracellular folding of newly synthesized or denatured proteins. Current evidence suggests an emerging role for hsp70 family members in immune responses and in clinically important responses to stress and tissue damage. Here we report the generation and characterization of several MAbs to hsp70 family members. Immune responses to this highly conserved family were induced in mice by immunization with synthetic peptides that contain regions of the mouse mitochondrial hsp70 coupled to a potent helper T cell epitope derived from tetanus toxoid. The resulting MAbs include ones specific for the human and mouse mitochondrial hsp70 and others that show cross-reactivity among the family members and recognize the mitochondrial hsp70, the endoplasmic reticulum resident hsp70, Bip/grp78, the constitutively expressed cytosolic hsp70, hsc70, and the heat-induced member, hsp70. Significantly, these MAbs are effective in Western blotting, in immunoprecipitation, and in immunofluorescence, and thus should find applications in the purification and detection of members of this important family.
Amino Acid Sequence↗
Chlamydia trachomatis RNA polymerase alpha subunit: sequence and structural analysis.
We describe the cloning and sequence analysis of the region surrounding the gene for the alpha subunit of RNA polymerase from Chlamydia trachomatis. This region contains genes for proteins in the order SecY, S13, S11, alpha, and L17, which are equivalent to Escherichia coli and Bacillus subtilis r proteins. The incorporation of chlamydial alpha subunit protein into the E. coli RNA polymerase holoenzyme rather than its truncated variant lacking the amino terminus suggests the existence of structural conservation among alpha subunits from distantly related genera.
Amino Acid Sequence↗
Gene-specific signal transduction between microtubules and tubulin genes in Tetrahymena thermophila.
Mammalian cells regulate tubulin mRNA abundance by a posttranscriptional mechanism dependent on the concentration of tubulin monomer. Treatment of mammalian cells with microtubule-depolymerizing drugs and microtubule-polymerizing drugs causes decreases and increases in tubulin mRNA, respectively (D. W. Cleveland, Curr. Opin. Cell Biol. 1:10-14, 1989). In striking contrast to the case with mammalian cells, perturbation of microtubules in Tetrahymena thermophila by microtubule-depolymerizing or -polymerizing drugs increases the level of the single alpha-tubulin gene message by increasing transcription (L. A. Stargell, D. P. Heruth, J. Gaertig, and M. A. Gorovsky, Mol. Cell. Biol. 12:1443-1450, 1992). In this report we show that antimicrotubule drugs preferentially induce the expression of one of two beta-tubulin genes (BTU1) in T. thermophila. In contrast, deciliation induces expression of both beta-tubulin genes. Tubulin gene expression was examined in a mutant strain created by transformation with an in vitro-mutagenized beta-tubulin gene that conferred resistance to microtubule-depolymerizing drugs and sensitivity to the polymerizing drug taxol and in a strain containing a nitrosoguanidine-induced mutation in the single alpha-tubulin gene that conferred the same pattern of drug sensitivities. In both cases the levels of tubulin mRNA expression from the drug-inducible BTU1 gene in the mutant cells paralleled the altered growth sensitivities to microtubule drugs. These studies demonstrate that T. thermophila has distinct, gene-specific mechanisms for modulating tubulin gene expression depending on whether ciliary or cytoplasmic microtubules are involved. They also show that the cytoplasmic microtubule cytoskeleton itself participates in a signal transduction pathway that regulates specific tubulin gene transcription in T. thermophila.
Animals↗
Transforming growth factor beta 1 messenger RNA in Reed-Sternberg cells in nodular sclerosing Hodgkin's disease.
AIMS: To determine the cellular origin of the most potent cytokine present in Hodgkin's disease, transforming growth factor (TGF) beta, the polycellular population of Hodgkin's tissue was studied using in situ hybridisation. METHODS: A biotin labelled oligo-complementary DNA (cDNA) was constructed according to the previously determined sequence for TGF beta 1 cDNA. Forty three frozen and paraffin wax embedded tissue samples replaced by Hodgkin's disease or non-Hodgkin's lymphoma, three Reed-Sternberg cell lines, one Ki1 positive lymphoma cell line, and an epithelial cell line were studied for expression of TGF beta 1 messenger RNA (mRNA) as well as secretion of the TGF beta 1 protein and expression of the CD30 epitope. RESULTS: The results obtained with the 24 frozen tissue samples confirmed that the TGF beta antigen is found predominantly in the nodular sclerosing Hodgkin's disease (NSHD) subtype. Nineteen paraffin wax embedded tissue samples were used to measure the simultaneous expression of CD30 and TGF beta 1 mRNA. The latter was found in eight of eight NSHD samples, two of six mixed cellularity samples, and two of five non-Hodgkin's lymphoma samples. No evidence of fibroblast expression of TGF beta 1 mRNA was noted. CONCLUSIONS: Activated lymphocytes in NSHD express TGF beta 1 mRNA, but binucleate Reed-Sternberg cells and mononuclear Hodgkin's cells are the primary sources of activated TGF beta in Hodgkin's disease.
Base Sequence↗
Homozygous deletions of the CDKN2 (MTS1/p16ink4) gene in cell lines established from children with acute lymphoblastic leukemia.
Homozygous deletions of the CDKN2 (MTS1/p16ink4) gene have been found at high frequency in cell lines derived from a variety of adult solid tumors. In order to investigate the status of the CDKN2 gene in cell lines established from childhood acute lymphoblastic leukemia (ALL), we surveyed 25 lines representing the major pediatric ALL phenotypes for the presence of this gene by Southern blot analysis. Homozygous deletions of all or part of the CDKN2 gene were detected in 21 (84%) cell lines, including 11 of 14 (79%) early-pre-B-ALL, four of five (80%) pre-B-ALL, and six of six T-ALL lines. CDN2 mRNA was detected by Northern blotting in each of the four lines containing an intact CDKN2 gene. These data suggest an important role for CDKN2 deletion in the cause and/or progression of pediatric ALL.
Blotting, Northern↗
V-J junctional sequences of T cell receptor gamma gene in acute lymphocytic leukemia.
T cell receptor TCR gamma gene rearrangement in a series of acute lymphocytic leukemia (ALL) patients was studied using PCR technique. 18 V-J junctional sequences (designated by N sequence) of TCR gamma gene were amplified with the V gamma and J gamma primers, using an unsymmetrical PCR and analysed by direct sequencing, demonstrating that in Chinese ALLs, the N sequences of TCR gamma gene are indeed clone-specific. Based on the known N sequences, several oligo-nucleotides were synthesized as probes, which were specific for the leukemic clone, and were used to detect the minimal residual disease (MRD) in 4 ALL cases. The sensitivity of this method was 0.1%-0.01%.
Adult↗
[Junctional sequences of T cell receptor V delta 2-D delta 3 or D delta 2-D delta 3 rearrangements in acute lymphoblastic leukemia].
T-cell receptor (TCR) delta chain gene rearrangements were studied by polymerase chain reaction (PCR) analysis in 46 patients with acute lymphoblastic leukemia (ALL). Sixteen patients were found to have incomplete rearrangements of the TCR delta genes. Among them, 13 patients displayed V delta 2-D delta 3 rearrangement, while 3 had both V delta 2-D delta 3 and D delta 2-D delta 3 rearrangements. To determine the junctional sequence of TCR delta gene, PCR products from the 16 patients were sequenced directly or after M13 cloning. The results showed the junctional sequences of TCR delta gene are highly specific for each allele. This sequence diversity resulted from several factors including deletion of the 3' end of V delta 2 or D delta 2 segment and 5' end of D delta 3 segment, the presence of D delta 1 or D delta 2 sequences, insertion of N nucleotides and the association of P nucleotides with intact V delta 2 and D delta 3 segments. In addition, analysis of N-nucleotide contents revealed that the amount of GC was much larger than that of AT (70%: 30%), indicating the insertion of N nucleotide was not fully random. Our sequence data confirmed that the imcomplete rearrangement of TCR delta gene is an early event in the lymphoid cell ontogenesis, and its N sequences in V-(D)-J junctional region may be used as a specific marker of clonality to detect the minimal residual disease (MRD) in ALL.
Adolescent↗
[Different patterns of follicular development and luteal function].
OBJECTIVE: To study the relationship between luteal function and different patterns of follicular development. METHODS: Different patterns of follicular development as observed under transvaginal B-ultrasound in 85 infertile patients included: (1) Ovulation from normal follicle (ONF). (2) Ovulation from two follicles at the same cycle (O2F). (3) Ovulation from small follicle (OSF). (4) Ovulation from polycystic ovaries (PCO). (5) Luteinized unruptured follicle syndrome (LUFS). (6) Anovulation with follicular atresia. The luteal function of the first 5 types was evaluated by serum and peritoneal fluid estrogen, progesterone levels, endometrial histology, estradiol receptor (ER) and progesterone receptor (PR) analyaes, and the length of luteal phase. RESULTS: The incidence of luteal phase deficiency (LPD): short luteal phase was 10.42%, retardation of endometrial development was 4.17% in ONF, no LPD in O2F, 21.42% in OSF, 2/3 in PCO, 3/6 in LUFS, respectively. The incidence of LPD was 14.58% in ONF, 56.52% in abnormal follicular development groups (OSF, O2F and LUFS) (P < 0.005). It was found that the retardation of endometrial development was associated either with lowered serum P level or normal serum P but lowered ER and PR concentrations. The latter condition was recognized as "pseudocorpus luteum deficiency". CONCLUSIONS: The different patterns of follicular development may exert a great influence over luteal function through the different production of steroid hormones and their receptors.
Corpus Luteum↗
Single amino acid changes alter the repair specificity of Drosophila Rrp1. Isolation of mutants deficient in repair of oxidative DNA damage.
Drosophila Rrp1 has several tightly associated enzymatic activities, including double-strand DNA 3'-exonuclease, apurinic/apyrimidinic endonuclease, 3'-phosphatase, and 3'-phosphodiesterase. The carboxyl-terminal third of Rrp1, homologous to Escherichia coli exonuclease III, is sufficient to repair oxidative and alkylation-induced DNA damage in vivo. Using a screen for partial complementation of repair-deficient E. coli, we isolated three mutants of the nuclease domain of Rrp1: T462A, K463Q, and L484P, that protect against methyl methanesulfonate (MMS)-induced but not t-BuO2H-induced DNA damage. Thr-462 and Lys-463 are highly conserved residues found in a cluster of 5 conserved amino acids (LQETK), while Leu-484 is poorly conserved. Gln-460 Glu-461, Thr-462, and Lys-463 and Leu-484 were altered by site-directed mutagenesis using a plasmid including the entire Rrp1 gene and mutant proteins were purified. Mutants of the three residues Glu-461, Thr-462, and Lys-463 demonstrate 8-200-fold lower phosphodiesterase specific activity than wild-type Rrp1. E461A has a 30-fold reduction in AP endonuclease and is MMS-sensitive, but all other mutants have near-normal AP endonuclease and are MMS-resistant. Glu-461 appears to be essential for the nuclease function for Rrp1. Lys-463 and, to a lesser extent, Thr-462 influence the substrate specificity of the Rrp1 nuclease.
Amino Acid Sequence↗
High frequency vector-mediated transformation and gene replacement in Tetrahymena.
Recently, we developed a mass DNA-mediated transformation technique for the ciliated protozoan Tetrahymena thermophila that introduces transforming DNA by electroporation into conjugating cells. Other studies demonstrated that a neomycin resistance gene flanked by Tetrahymena H4-I gene regulatory sequences transformed Tetrahymena by homologous recombination within the H4-I locus when microinjected into the macronucleus. We describe the use of conjugant electrotransformation (CET) for gene replacement and for the development of new independently replicating vectors and a gene cassette that can be used as a selectable marker in gene knockout experiments. Using CET, the neomycin resistance gene flanked by H4-I sequences transformed Tetrahymena, resulting in the replacement of the H4-I gene or integrative recombination of the H4-I/neo/H4-I gene (but not vector sequences) in the 5' or 3' flanking region of the H4-I locus. Gene replacement was obtained with non-digested plasmid DNA but releasing the insert increased the frequency of replacement events about 6-fold. The efficiency of transformation by the H4-I/neo/H4-I selectable marker was unchanged when a single copy of the Tetrahymena rDNA replication origin was included on the transforming plasmid. However, the efficiency of transformation using CET increased greatly when a tandem repeat of the replication origin fragment was used. This high frequency of transformation enabled mapping of the region required for H4-I promoter function to within 333 bp upstream of the initiator ATG. Similarly approximately 300 bp of sequence downstream of the translation terminator TGA of the beta-tubulin 2 (BTU2) gene could substitute for the 3' region of the H4-I gene. This hybrid H4-I/neo/BTU2 gene did not transform Tetrahymena when subcloned on a plasmid lacking an origin of replication, but did transform at high frequency on a two origin plasmid. Thus, the H4-I/neo/BTU2 cassette is a selectable marker that can be used for gene knockout in Tetrahymena. As a first step toward constructing a vector suitable for cloning genes by complementation of mutations in Tetrahymena, we also demonstrated that the vector containing 2 origins and the H4-I/neo/BTU2 cassette can co-express a gene encoding a cycloheximide resistant ribosomal protein.
Animals↗
The genome reconstruction manager: a software environment for supporting high-throughput DNA sequencing.
A new software system designed for use in high-throughput DNA sequencing laboratories is described. The Genome Reconstruction Manager (GRM) was developed from requirements derived from ongoing large-scale DNA sequencing projects. Object-oriented principles were followed in designing the system, and tools supporting object-oriented system development were employed for its implementation. GRM provides several advances in software support for high-throughput DNA sequencing: support for random, directed, and mixed sequencing strategies; a novel system for fragment assembly; a commercial object data-base management system for data storage; a client/server architecture for using network computational servers; and an underlying data model that can evolve to support fully automatic sequence reconstruction. GRM is currently being deployed for production use in high-throughput DNA sequencing projects.
Algorithms↗
Transcription factor GATA-2 is expressed in erythroid, early myeloid, and CD34+ human leukemia-derived cell lines.
To understand the functional roles that the GATA factors may play during hematopoietic cell differentiation, we examined the expression of GATA factor mRNAs and protein products in various human cell lines. Blot hybridization analyses demonstrated that GATA-1 and GATA-2 mRNAs are expressed abundantly in a set of cell lines established from human myelogenous leukemia cells, but the expression pattern of each factor is distinct. GATA-2 mRNA is expressed in all cell lines tested that express erythroid markers, and, in addition, the mRNA is also expressed in three CD34+ cell lines and two early myeloid cell lines. In contrast, the expression of GATA-1 mRNA showed tight correlation to that of the erythroid/megakaryocytic lineage markers. We also found that the GATA-2 probe identifies two types of mRNA. Structural analysis of genomic DNA clones encoding human GATA-2 coupled with RNA blot analysis demonstrated that there exists an alternative use of polyadenylation consensus sequences in a single exon and this causes the molecular heterogeneity among GATA-2 mRNAs. Through immunochemical and immunohistochemical analyses using anti-GATA-1- and anti-GATA-2-specific antibodies, GATA-2 protein was clearly shown to be present in the nuclei of leukemia-derived early myeloid and CD34+ cell lines, whereas both GATA-1 and GATA-2 proteins are expressed in erythroid/megakaryocytic cell lines. Thus, the expression profile of GATA-2 is consistent with the hypothesis that GATA-2 plays unique roles for the transcriptional activation of genes in cells at an early stage of hematopoietic differentiation and in developing cells of the erythroid and myeloid lineages.
Antibodies, Monoclonal↗
Influence of hydrocarbon tail structure on quinone binding and electron-transfer performance at the QA and QB sites of the photosynthetic reaction center protein.
Binding free energies of 37 functional replacement quinone cofactors with systematically altered hydrocarbon tail structures have been determined for the QA and QB redox catalytic sites of the reaction center protein isolated from Rhodobacter sphaeroides and solubilized in aqueous and in hexane solutions. The first two and part of the third isoprene units of the 10-unit tail of the native ubiquinone-10 cofactor interact with the protein interior at each site. Contributions of the same tail structures to the binding free energies of quinones at the QA and QB sites are comparable, suggesting that the binding domains share common features. Comparison of the affinities of a homologous series of 10 n-alkyl-substituted ubiquinones resolves the binding forces along the length of the tail binding domain and shows that strong steric constraints oppose accommodation of the tail in its extended conformation. Differences in the contributions of identical tail substituents to ubiquinone- and menaquinone-QA site affinities, and tail-induced changes of up to 5-fold in the rates of QA site-mediated electron-transfer reactions, suggest that the tail adjusts the position of the quinone ring. Substitution of ubiquinone with the native 10-unit isoprene tail does not alter the affinity for the sites as determined in hexane solution. However, one- and two-isoprene-substituted quinones bind more tightly than analogs substituted with saturated-alkyl tail substituents. The sites therefore exhibit binding specificity for the native isoprene tail structure. Calculations indicate that the binding specificity arises primarily from a lower integrated torsion potential energy in the bound isoprene tails. The results suggest that the in vivo tail-protein interaction is designed to deter competitive interference of quinone function by amphiphilic species present in the native membrane.
Crystallography, X-Ray↗
Cloning and characterization of a secY homolog from Chlamydia trachomatis.
Characterization of the genes involved in the process of protein translocation is important in understanding their structure-function relationships. However, little is known about the signals that govern chlamydial gene expression and translocation. We have cloned a 1.7 kb HindIII-PstI fragment containing the secY gene of Chlamydia trachomatis. The complete nucleotide sequence reveals three open reading frames. The amino acid sequence shows highest homology with Escherichia coli proteins L15, SecY and S13, corresponding to the spc-alpha ribosomal protein operons. The product of the C. trachomatis secY gene is composed of 457 amino acids with a calculated molecular mass of 50,195 Daltons. Its amino acid sequence shows 27.4% and 35.7% identity to E. coli and Bacillus subtilis SecY proteins, respectively. The distribution of hydrophobic amino acids in the C. trachomatis secY gene product is suggestive of it being an integral membrane protein with ten transmembrane segments, the second, third and seventh membrane segments sharing > 45% identity with E. coli SecY. Our results suggest that despite evolutionary differences, eubacteria share a similar protein export apparatus.
Amino Acid Sequence↗
Electroporation-mediated replacement of a positively and negatively selectable beta-tubulin gene in Tetrahymena thermophila.
Replacement of lysine-350 by methionine in the beta-tubulin gene of Chlamydomonas confers resistance to microtubule-depolymerizing drugs and increased sensitivity to the microtubule-stabilizing drug taxol. This mutation was created in cloned BTU1, one of two coexpressed beta-tublin genes of Tetrahymena thermophila. When introduced by electroporation, the mutated gene transformed Tetrahymena exclusively by gene replacement at the homologous locus. Taxol-sensitive transformants could be retransformed with a wild-type gene and selection for taxol resistance. Analyses of phenotypic assortment and of the mRNA in transformed cells suggest that complete replacement of the BTU1 gene in the polyploid macronucleus can be obtained. These studies demonstrate the utility of this marker for studying tublin gene function and show that electroporation allows facile gene replacement in Tetrahymena.
Animals↗
Three microsatellites defining four alleles for the rat SA gene.
Explore the source record for details and available documents.
Alleles↗