The interaction trap: in vivo analysis of protein-protein associations.
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Arabidopsis↗
Biomedical subjects
I Hwang
Publications and source records attributed to I Hwang.
Solid solubilities of magnetic ions in diluted magnetic semiconductors grown under equilibrium conditions.
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Journal Article↗
TraI, a LuxI homologue, is responsible for production of conjugation factor, the Ti plasmid N-acylhomoserine lactone autoinducer.
Conjugal transfer of the nopaline-type Agrobacterium Ti plasmid pTiC58 is regulated by a transcriptional activator, TraR, and a diffusible signal molecule, conjugation factor (CF). CF is a member of a family of substituted homoserine lactones (HSLs) that act as coinducers for regulating gene expression in diverse Gram-negative bacteria by a mechanism called autoinduction. In Vibrio fischeri HSL production is conferred by the luxI gene. Homologues of this gene are responsible for HSL production by other Gram-negative bacteria. A gene that we call traI, conferring production of material with CF activity, was localized to a 1-kb region at the upstream end of tra3 of pTiC58. Spectroscopy showed that the activity was authentic CF. Sequence analysis showed that traI could encode a protein of 211 amino acids, TraI, that is related to the proteins responsible for HSL production by other bacteria. A second, partial open reading frame immediately downstream of traI could encode a protein related to TrbB of plasmid RP4, which is required for conjugal transfer. Transcription of traI and of the downstream tra3 genes requires TraR and CF and initiates from the traI promoter. The results show that traI is responsible for CF production, that it is the first gene of the tra3 operon, and that expression of this operon is regulated by autoinduction.
Amino Acid Sequence↗
Isolation and characterization of two beta-tubulin cDNA clones from rice.
Two cDNA clones encoding two different beta-tubulins, RTUB-1 and RTUB-2, were isolated from a rice cDNA library and their nucleotide sequences were analyzed. The deduced amino acid sequences showed amino acid sequence identity between 92% and 97% with other plant beta-tubulins. Southern blot analysis using gene-specific and coding-region probes suggested that beta-tubulins in rice are encoded by multigene families. The two cDNA clones represent two subfamilies of rice tubulins, RTUB-1 and RTUB-2, consisting of 3 to 4 genes and a single gene, respectively. The transcript levels of RTUB-1 and RTUB-2 genes were higher in actively elongating tissues such as etiolated shoot tissues and light-grown root tissues of four-day old seedlings.
Amino Acid Sequence↗
Cis-acting DNA elements and cell type-specific nuclear proteins which may play a role in regulation of mouse CD8 alpha (Lyt-2) gene transcription.
Fusion of mouse CD8+ class I MHC-restricted T cells with the BW5147 thymoma invariably yields CD8- hybridomas in which RNA transcribed from the CD8 alpha (Lyt-2) gene is undetectable. To determine whether cis-acting DNA sequences may negatively regulate transcription of the Lyt-2 gene in BW5147 cells, one possible explanation for the above observation, BW5147 cells were stably transfected with the Lyt-2 gene containing 1 - 11,000 nucleotides of 5' flanking DNA and surface expression of Lyt-2 was monitored by flow microfluorometry. Initial results suggested the presence of a negative element between 1400 and 5000 nucleotides upstream of the site of transcription initiation. Further studies suggested the presence of two potential negative regulatory elements in this region, one of which includes a 269 nucleotide Accl - SstI fragment comprised of nucleotides -4700 to -4431 which bound nuclear proteins from CD8+ and CD8- cell lines in electrophoretic mobility shift assays (EMSA). EMSA studies performed using nuclear extracts from a variety of cell lines and tissues demonstrated that unique retarded complexes, called bands 1 and 2, correlated significantly with expression or non-expression of Lyt-2 respectively. EMSA analysis of proteins fractionated by SDS-PAGE from nuclear extract of the CD8+ VL3 T lymphoma cell line revealed proteins of approximately 110-130 kDa (called L2a-P1) and > 200 kDa (called L2a-P2) which bind within a 100 nucleotide region of this fragment (called L2a) to yield band 1 and 2 respectively, and which may play a role in regulation of Lyt-2 gene transcription.
Animals↗
A novel gene tag for identifying microorganisms released into the environment.
A novel method using a moc (mannityl opine catabolism) region from the Agrobacterium tumefaciens Ti plasmid pTi15955 was developed as a tag to identify genetically modified microorganisms released into the environment. Pseudomonas fluorescens 1855.344, a plant-growth-promoting rhizosphere bacterium, was chosen as the organism in which to develop and test the system. moc genes carried by pYDH208, a cosmid clone containing a 20-kb segment of the octopine-mannityl opine-type Ti plasmid, conferred on P. fluorescens strains the capacity to utilize mannopine and agropine (AGR) as a sole source of carbon and energy. Modified P. fluorescens strains containing moc or moc::nptII inserted into a chromosomal site were constructed by marker exchange. One such modified strain, PF5MT12, utilized AGR as a sole carbon source and contained detectable levels of mannopine cyclase, an easily assayable enzyme encoded by the moc region. Catabolism of AGR could be used to recover selectively the marked strain from mixed populations containing a large excess of closely related bacteria. Nucleic acid-based detection strategies were developed on the basis of the unique fusion region between Agrobacterium DNA and Pseudomonas DNA in strain PF5MT12. The specificity and sensitivity of detection of PF5MT12 were enhanced by amplifying the fused DNA region by using PCR. The target fragment could be detected at levels of sensitivity comparable to those of other described PCR-based gene tags, even in the presence of high levels of Agrobacterium, Pseudomonas, or Escherichia coli DNA. This gene tag strategy gives a method for direct selection and enumeration of the marked strain from mixtures containing a large excess of closely related bacteria and a sensitive and highly specific system for detection by PCR amplification of the target fragment even in the presence of large amounts of DNA from related or unrelated organisms.
Agrobacterium tumefaciens↗
Differential susceptibility of mouse Lyt-2 and Lyt-3 genes to negative regulation.
Hybrids of Lyt-2/Lyt-3-positive class I-specific cytotoxic T lymphocytes (CTLs) with the BW5147 thymoma cell line (Lyt-2/Lyt-3-negative) are known to be Lyt-2/Lyt-3-negative due to shutoff of transcription of the CTL's Lyt-2 gene. Hybrids of a constitutively Lyt-2-positive transfectant of BW5147 (3B2) with a long term CTL line, 2C, and with CTLs generated in a mixed leucocyte reaction (MLR) shut off the CTL's Lyt-2 gene as expected but express the CTL's Lyt-3 gene product as a heterodimer with the product of the transfected Lyt-2 gene. Thus the Lyt-3 gene is not subject to the same negative regulatory influences as the Lyt-2 gene. That expression of Lyt-2 is not necessary for Lyt-3 gene transcription to continue is demonstrated by the finding that hybrids of MLR-generated CTLs with either BW5147 (Lyt-2-negative) or 3B2 (Lyt-2-positive) cells express Lyt-3 RNA. Southern hybridization and structural analysis of DNA fragments generated using the polymerase chain reaction demonstrated that hybrids contain several species of Lyt-3 RNA, one of which lacks the exon encoding the extracellular V-like domain and appears to be the product of an alternatively-spliced RNA transcript.
Animals↗
Cloning of an Arabidopsis ribosomal protein S28 cDNA.
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Arabidopsis↗
Map-based cloning of a gene controlling omega-3 fatty acid desaturation in Arabidopsis.
A gene from the flowering plant Arabidopsis thaliana that encodes an omega-3 desaturase was cloned on the basis of the genetic map position of a mutation affecting membrane and storage lipid fatty acid composition. Yeast artificial chromosomes covering the genetic locus were identified and used to probe a seed complementary DNA library. A complementary DNA clone for the desaturase was identified and introduced into roots of both wild-type and mutant plants by Ti plasmid-mediated transformation. Transgenic tissues of both mutant and wild-type plants had significantly increased amounts of the fatty acid produced by this desaturase.
Amino Acid Sequence↗
The Monitored Atherosclerosis Regression Study (MARS). Design, methods and baseline results.
OBJECTIVE: The Monitored Atherosclerosis Regression Study (MARS) was designed to evaluate the effect of cholesterol lowering by monotherapy with an HMG-CoA reductase inhibitor on progression/regression of atherosclerosis in subjects with angiographically documented coronary artery disease. The purpose of this paper is to present the design, methods, and baseline results of MARS. DESIGN: MARS is a prospective, randomized, double-blind, placebo-controlled trial with baseline, 2-year, and 4-year coronary angiography as well as carotid, brachial, and popliteal ultrasonography. SETTING: Outpatient clinics at the University of Southern California School of Medicine and the University of Wisconsin School of Medicine. SUBJECTS: Two hundred seventy participants of both sexes were recruited directly from the cardiac catheterization laboratory or by chart review of patients having undergone cardiac catheterization in the past. Subjects were considered eligible if they had angiographically demonstrable atherosclerosis in 2 or more coronary artery segments, unaltered by angioplasty, with at least 1 lesion > or = 50% but < 100% diameter stenosis (%S). The inclusion range for total cholesterol (TC) was between 190 and 295 mg/dL. Exclusion factors were: triglycerides > or = 500 mg/dL; premenopausal females; uncontrolled hypertension; diabetes mellitus; untreated thyroid disease; liver dysfunction; renal insufficiency; congestive heart failure; major arrhythmia; left ventricular conduction defects; or any life-threatening disease. INTERVENTION: Subjects were placed on a low-fat, low-cholesterol diet and either 40 mg b.i.d. lovastatin (Mevacor) or placebo. Randomization was stratified by sex, smoking status, and TC. MAIN OUTCOME MEASURES: Per-subject average change in %S as determined by quantitative coronary angiography (QCA) is the primary angiographic endpoint. Secondary endpoints are: categorical analyses of the proportion of subjects with progression; human panel reading of coronary angiograms; and change in minimum lumen diameter (MLD) in mm by QCA. Carotid, brachial, and popliteal ultrasonography is also being performed. RESULTS: The subjects randomized into MARS are 91.5% male with an age range of 37 to 67 years (mean age 57.9 years). For the cohort, baseline lipids are (mean +/- SD): TC, 231 +/- 24 mg/dL; low-density lipoprotein cholesterol (LDL-C) by ultracentrifugation, 153 +/- 24 mg/dL; LDL-C, by calculation, 157 +/- 23 mg/dL; high-density lipoprotein cholesterol (HDL-C), 43 +/- 10 mg/dL; and triglycerides, 160 +/- 73 mg/dL. There were no significant differences between treatment groups in baseline lipid levels or baseline angiographic characteristics. CONCLUSIONS: MARS baseline data show adequacy of randomization with comparability of lovastatin and placebo groups in demographic, lipid, and angiographic characteristics.
Adult↗
Cloning and characterization of pathogenicity genes from Xanthomonas campestris pv. glycines.
Nonpathogenic mutants of Xanthomonas campestris pv. glycines 8ra were generated with N-methyl-N-nitro-N'-nitrosoguanidine to identify and characterize pathogenicity genes of the bacterium. A total of 16 nonpathogenic mutants were isolated from 2,000 colonies. One mutant, NP1, was chosen for further study. NP1 did not multiply in soybean cotyledons. A genomic library of strain 8ra was constructed in the cosmid pLAFR3, and the cosmids were tested for complementation in NP1. One cosmid clone, pIH1, which contained a 31-kb insert, complemented mutant NP1. A restriction map of pIH1 was constructed, and deletion analyses identified a 10-kb HindIII fragment that restored pathogenicity to NP1. Southern hybridization analysis indicated that DNA sequences in the 10-kb HindIII fragment are conserved among other X. campestris pathovars tested. Three regions responsible for restoring pathogenicity have been identified by Tn3-HoHo1 mutagenesis. A 2.7-kb ClaI fragment was sequenced, and two possible open reading frames (ORF1 and ORF2) were found. Results indicated that ORF2 but not ORF1 may be expressed in Escherichia coli and in X. campestris pv. glycines. The carboxy terminus of the potential polypeptide encoded by ORF2 has an amino acid sequence similar to that of the gamma subunit of oxaloacetate decarboxylase, which is involved in sodium ion transport in Klebsiella pneumoniae.
Amino Acid Sequence↗
Identification and map position of YAC clones comprising one-third of the Arabidopsis genome.
YAC clones corresponding to 125 Arabidopsis thaliana RFLP markers have been identified. At least one YAC clone has been isolated for each of the RFLP markers tested. Based on CHEF gel analysis of 196 clones, the mean insert size of the available Arabidopsis YAC libraries is approximately 160 kb. The YACs of known genetic map location encompass about 30% of the Arabidopsis genome. The results presented here represent a first step towards assembly of an overlapping YAC library of the A. thaliana genome.
Arabidopsis↗
S-phase-specific transcription regulatory elements are present in a replication-independent testis-specific H2B histone gene.
The testis-specific H2B histone (TH2B) gene is expressed in pachytene spermatocytes during meiotic prophase I in the absence of any significant DNA synthesis. Unlike somatic histones, synthesis of testis-specific histones is not affected by inhibitors of DNA synthesis. A genomic rat TH2B gene was cloned by using a DNA fragment derived from TH2B cDNA as a probe. Expression of the cloned TH2B was investigated by gene transfer experiments. From these studies, we found that the 5' upstream region of the cloned TH2B gene contained S-phase-specific transcription elements which regulated expression of a reporter gene in an S-phase-specific manner. The S-phase-regulatory element was found to be located in two regions containing CCAAT elements between -153 and -110 base pairs (bp) and an octamer element (ATTTGCAT) between -109 and -84 bp. The two regions were required for a maximal stimulation of transcription of the cloned TH2B gene in S phase. On the other hand, only the octamer element was reported be important for the S-phase-specific transcription of human H2B gene. Since the synthesis of the TH2B histone is independent of DNA synthesis and specific for pachytene spermatocytes in vivo, the presence of the S-phase-specific transcription regulatory elements in the TH2B gene is surprising.
Animals↗
Molecular cloning and differential expression of somatic and testis-specific H2B histone genes during rat spermatogenesis.
We have cloned cDNA of a testis-specific histone, TH2B (a variant of H2B), and rat somatic H2B gene to investigate regulation of testis-specific histone genes during rat spermatogenesis. The amino acid sequences deduced from DNA sequences show extensive sequence divergence in the N-terminal third of the two histones. The rest is highly conserved. One cysteine residue was found in TH2B. No cysteine is present in somatic histones except in H3 histone. We investigated the expression of TH2B and H2B genes using the regions of sequence divergence as hybridization probes. The TH2B gene is expressed only in the testis, and the expression of this gene is detected 14 days after birth, reaching a maximum at Day 20. The level of H2B mRNA shows a reciprocal pattern. This contrasting pattern can be explained by the gradually changing proportion of spermatogonia and spermatocytes with testicular maturation. In situ cytohybridization studies show that H2B gene is expressed primarily in proliferating spermatogonia and preleptotene spermatocytes, whereas TH2B gene is expressed exclusively in pachytene spermatocytes which first appear in testis about 14 days after birth. H2B and TH2B genes appear to be ideal markers for the study of proliferation and differentiation events in spermatogenesis and their regulatory mechanisms.
Amino Acid Sequence↗
The superior and inferior components of Whitnall's ligament.
During an orbital dissection in a 60-year-old white male, we came across a very well defined superior and inferior component of Whitnall's ligament (Figure 1). The superior component of Whitnall's ligament is well described and well known to ophthalmologists performing eyelid surgery. The inferior component, however, is not well recognized or described. This cadaver dissection illustrates both Whitnall's components well.
Cadaver↗