YY1 is a multifunctional transcription factor capable of either activation or repression of transcription. Using a series of mutant proteins, we have characterized domains responsible for activation or repression. We found that the YY1 transcriptional activation domain lies near the amino terminus and requires amino acids 16-29 and 80-100 for maximal activity. The region between residues 16 and 29 has the potential to form an acidic amphipathic helix, whereas residues between 80 and 100 are rich in proline and glutamine. The YY1 repression domain lies near the carboxyl terminus and is embedded within the YY1 zinc finger region necessary for binding to DNA. Deletion of YY1 amino acids, which include zinc fingers 3 and 4, abolishes repression. However, site-directed mutagenesis, progressive deletion, and internal deletion mutant analyses indicate that the normal structures of zinc fingers 3 and 4 are not required for repression.

3T3 Cells↗

PubMed1995

A transforming growth factor beta receptor type II gene mutation common in colon and gastric but rare in endometrial cancers with microsatellite instability.

We have recently demonstrated that mutation of the transforming growth factor-beta (TGF-beta) receptor type II (RII) gene is characteristic of colon cancers exhibiting microsatellite instability or replication errors (RER+). Moreover, we have shown that RII mutations in these RER+ colon cancers are characteristically frameshift mutations within a 10-bp polyadenine repeat present in the RII-coding region. We now show that RII gene mutations in this polyadenine repeat are also commonly present in RER+ gastric cancers (71%). In contrast, we find these same RII gene mutations are distinctly uncommon in RER+ endometrial cancers (17%, P < 0.02). These results suggest that RII gene mutations confer a growth advantage and are selected for in RER+ cancers of both the upper and lower gastrointestinal tract. The genesis of RER+ endometrial tumors must, however, be by a different route.

Base Sequence↗

PubMed1995

A domain of the gene 4 helicase/primase of bacteriophage T7 required for the formation of an active hexamer.

The bacteriophage T7 gene 4 protein, like a number of helicases, is believed to function as a hexamer. The amino acid sequence of the T7 gene 4 protein from residue 475 to 491 is conserved in the homologous proteins of the related phages T3 and SP6. In addition, part of this region is conserved in DNA helicases such as Escherichia coli DnaB protein and phage T4 gp41. Mutations within this region of the T7 gene 4 protein can reduce the ability of the protein to form hexamers. The His475-->Ala and Asp485-->Gly mutant proteins show decreases in nucleotide hydrolysis, single-stranded DNA binding, double-stranded DNA unwinding, and primer synthesis in proportion to their ability to form hexamers. The mutation Arg487-->Ala has little effect on oligomerization, but nucleotide hydrolysis by this mutant protein is inhibited by single-stranded DNA, and it has a higher affinity for dTTP, suggesting that this protein is defective in the protein-protein interactions required for efficient nucleotide hydrolysis and translocation on single-stranded DNA. Gene 4 protein can form hexamers in the absence of a nucleotide, but dTTP increases hexamer formation, as does dTDP, to a lesser extent, demonstrating that the protein self-association affinity is influenced by the nucleotide bound. Together, the data demonstrate that this region of the gene 4 protein is important for the protein-protein contacts necessary for both hexamer formation and the interactions between the subunits of the hexamer required for coordinated nucleotide hydrolysis, translocation on single-stranded DNA, and unwinding of double-stranded DNA. The fact that the gene 4 proteins form dimers, but not monomers, even while hexamer formation is severely diminished by some of the mutations, suggests that the proteins associate in a manner with two separate and distinct protein-protein interfaces.

Amino Acid Sequence↗

PubMed1995

Urinary sediment analyzed by flow cytometry.

A flow cytometer for the automated analysis of urinary sediment was designed, and its performance was examined by the evaluation of 821 specimens. Auramine O, a dye for DNA and RNA, was used for the staining of the sediment. Urine (5 ml or more) was processed by the instrument for sediment analysis. Conventional microscopic analysis was done for comparison. The RBC count, the WBC count, and the number of bacterial cells, epithelial cells, and casts found by the flow cytometer and by microscopy were compared. Correlation was high for all these results. The overall sensitivity, specificity, and efficiency (accuracy) in the items analyzed were 84.7%, 57.8%, and 67.2%, respectively. One hundred specimens could be analyzed by the instrument per hour. The instrument seemed useful for screening for urinary tract disorders to identify specimens that should be analyzed microscopically in routine laboratories.

Adolescent↗

PubMed1995

(1 beta, 5 alpha, 6 beta)-10,10- (1,2-ethylenedioxy)-1,5-dimethylbicyclo[4.4.0]decan-4-one.

The title compound (1) [alternative name: 5,8a-dimethyl(decahydronaphthalene)-1-spiro-2'-(1',3'-dioxolan+ ++)-6-one, C14H22O3] was obtained as the major product upon Pd/CaCO3-catalyzed hydrogenation of the corresponding enone (2). The analysis of the crystal structure of (1) has established that the monoacetal is cis-fused and that both rings adopt nearly perfect chair forms. The C(5) methyl group is in an alpha position, resulting directly from syn-hydrogenation of the enone. While there are two possible chair-chair conformations for cis-decalins, the title compound crystallized in the conformation which places the C(5) methyl group in an equatorial position.

Crystallography, X-Ray↗

PubMed1995

Mycoplasma virus P1 has a linear, double-stranded DNA genome with inverted terminal repeats.

Mycoplasma virus P1 is a tailed, polyhedral virus isolated from Mycoplasma pulmonis. To characterize the P1 genome, stocks of virus were prepared free of host cell nucleic acids. A single DNA species of 11.3 kb that was shown by plaque hybridization to be of P1 origin was extracted from the virus. Efficient isolation of P1 DNA required digestion with proteolytic enzymes prior to phenol extraction. Although P1 DNA was double-stranded and linear following such treatment, it was resistant to digestion with the 5'-specific lambda exonuclease. Electron microscopic analysis indicated that globular material is complexed to the ends of P1 DNA. The globular material was not observed on protease-treated P1 DNA molecules, suggesting that it is composed of protein. Removal of the putative terminal protein by chemical treatment allowed the cloning of the P1 DNA ends, and nucleotide sequence analysis revealed that these ends contain a 350-bp inverted terminal repeat.

Bacteriophages↗

PubMed1995

Chaperone SecB: conformational changes demonstrated by circular dichroism.

The chaperone SecB, which is involved in protein export in Escherichia coli, is shown by circular dichroism measurements to contain a high content of beta-pleated sheets. Prediction of the secondary structure of SecB is in good agreement with the observed content of beta-sheet. In accordance with the previous studies in which changes in conformation were assessed indirectly [Randall (1992), Science 257, 241-245], here we show that the conformation of SecB changes with the concentration of salt in the milieu and also when SecB interacts with a peptide ligand.

Bacterial Proteins↗

PubMed1995

Grafting of ethylene glycol-butadiene block copolymers onto dimethyl-dichlorosilane-coated glass by gamma-irradiation.

Amphipathic ethylene glycol-butadiene block copolymers (PEG-PB) with different chain lengths of poly(ethylene glycol) (PEG) were synthesized by reacting poly(ethylene glycol methyl ether) (m-PEG, mol. wt = 350, 550, 750, 2000 and 5000) with telechelic polybutadiene (PB). The PEG-PB copolymers formed were covalently grafted to dimethyldichlorosilane-coated glass (DDS-glass) by gamma-irradiation. The PEG-grafted surface was characterized by measuring advancing and receding contact angles, fibrinogen adsorption, the number of adherent platelets and the area of spread platelets. The grafting efficiency was measured indirectly from the ability of the surface to prevent platelet adhesion. The total dose of gamma-irradiation necessary for grafting of PEG-PB onto DDS-glass in aqueous solutions was less than 0.24 Mrad at atmospheric pressure and ambient temperature. For successful grafting, the surface-adsorbed copolymers should be gamma-irradiated in the presence of water. gamma-Irradiation in the dried state did not result in copolymer grafting. The adsorption of copolymers for 30 min before exposure to gamma-irradiation was enough for effective grafting. The grafting was equally effective whether or not DDS-glass was exposed to the air-copolymer solution interface when the DDS-glass was introduced into the copolymer solution. The copolymers were able to prevent platelet adhesion only when they were adsorbed onto DDS-glass at certain bulk concentrations. Too low or too high copolymer concentrations in the adsorption solution resulted in a surface where platelets could adhere and activate. The range of copolymer concentration which prevented platelet adhesion was larger as the PEG chain length of the grafted copolymers became longer. Our data indicate that platelet-resistant surfaces can be made by grafting PEG-PB onto chemically inert surfaces by a simple gamma-irradiation process.

Adsorption↗

PubMed1995

What is the basis of sequence-directed curvature in DNAs containing A tracts?

A variety of solution and gel experiments show that DNAs containing tracts of 4-8 A's repeated in phase with the helix repeat are curved. Several independent analyses of these experiments argue that curvature resides in the A tracts themselves. In x-ray crystallographic studies of several DNAs containing A tracts, however, the A tracts are uncurved, leading to models in which curvature resides in the non-A tracts. This "curved general sequence model" has several problems, in our view. We review those, and we describe recent experiments that show that the dehydrating agents commonly used in x-ray crystallography markedly reduce curvature in gels and in solution, calling into question the ability of crystallography to determine the structural basis of DNA curvature. Finally, we discuss the critical role of hydration in curved DNAs and suggest new experiments that we hope could finally determine exactly which sequences are responsible for curvature.

Adenine↗