bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of an inducible factor that binds to a positive regulatory element of the human beta-interferon gene.
Authors: Keller A D , Maniatis T
Published: May 2006

Human beta-interferon gene expression is induced by virus or poly(I).poly(C). This induction is due at least in part to an increase in the rate of transcription and does not require protein synthesis. A 40-base-pair DNA sequence within the beta-interferon promoter, termed the interferon gene regulatory element (IRE), is an inducible enhancer in mouse fibroblasts, and both positive and negative regulatory DNA sequences have been identified within this element. In this paper we identify three factors that bind specifically to two positive regulatory domains within the IRE. Two of these factors are present in nuclear extracts prepared from uninduced and induced cells; one is present only in extracts from induced cells. The functional significance of these binding activities was demonstrated by showing that point mutations within the IRE that decrease human beta-interferon gene transcription in vivo prevent binding in vitro. We propose that induction of the beta-interferon gene involves the modification of a protein to a form that binds specifically to a positive regulatory sequence within the IRE.
Cold denaturation of staphylococcal nuclease.
Authors: Griko Y V , Privalov P L , Sturtevant J M , Venyaminov SYu
Published: May 2006

Denaturation of staphylococcal nuclease was studied in a temperature range from -7 to 70 degrees C by scanning microcalorimetry and spectropolarimetry. It was found that the native protein is maximally stable at about 20 degrees C and is denatured upon heating and cooling from this temperature. The heat and cold denaturation processes are approximated rather well by a two-state transition showing that the molecule is composed of a single cooperative system. The main difference between these two processes is in the sign of the enthalpy and entropy of denaturation: whereas the heat denaturation proceeds with increases in the enthalpy and entropy, the cold denaturation proceeds with decreases in both quantities. The inversion of the enthalpy sign occurs at about 15 degrees C in an acetate buffer, but this temperature can be raised by addition of urea to the solvent.
Stabilization of an associated folding intermediate of bovine growth hormone by site-directed mutagenesis.
Authors: Brems D N , Plaisted S M , Havel H A , Tomich C S
Published: May 2006

By using oligonucleotide-directed mutagenesis, Lys-112 of bovine growth hormone (bGH) was changed to leucine, and its resulting effect on folding was studied. Equilibrium denaturation curves for the mutant protein exhibit biphasic or nonsymmetrical transitions by a variety of spectroscopic and hydrodynamic techniques, whereas the wild-type protein at the same concentration exhibits symmetrical transitions. The mutant protein refolds slower (by a factor of 30) and more readily precipitates upon refolding than the wild-type protein. These folding characteristics of the mutant protein are demonstrated to be a result of stabilization of an associated folding intermediate. A 38-amino acid fragment (96-133) derived from the mutant protein is helical, likely amphipathic, and more stabilized by increasing peptide concentration than is the corresponding helical peptide from the wild-type protein. The increased stability of the associated intermediate and the increased helicity of the peptide from the mutant protein are explained by preferential intermolecular interactions between helices due to enhanced hydrophobic attraction by their amphipathic surfaces.
A Xenopus ribosomal protein S6 kinase has two apparent kinase domains that are each similar to distinct protein kinases.
Authors: Jones S W , Erikson E , Blenis J , Maller J L , Erikson R L
Published: May 2006

We report the molecular cloning of cDNAs for S6 kinase II (S6KII) mRNAs present in Xenopus ovarian tissue. Two cDNAs were isolated by hybridization to oligonucleotide probes designed to encode tryptic peptides isolated from S6KII. The two cDNAs show 91% sequence similarity to each other. These two cDNAs predict proteins of 733 (S6KII alpha) and 629 (S6KII beta) amino acids that show 95% sequence similarity over the 629 amino acids where they are colinear. Amino acids 44-733 of S6KII alpha were expressed in Escherichia coli and the recombinant protein was used to raise antiserum in rabbits. This antiserum reacted with authentic S6KII prepared from Xenopus eggs. This interaction was specifically blocked by the recombinant protein from E. coli. The sequences of S6KII alpha and -beta predict four tryptic peptides whose sequences are identical to four peptides isolated from a tryptic digest of S6KII. The S6KII proteins have a very unusual structure when compared with previously studied protein kinases. They contain two apparent kinase domains, each similar to distinct protein kinases. The amino-terminal 366 amino acids show high sequence similarity to the regions of protein kinase C, the catalytic subunit of cAMP-dependent protein kinase, and cGMP-dependent protein kinase that contain the sites for ATP binding and are believed to be the catalytic centers for phosphotransferase activity. The remainder of the S6 kinase molecule shows high sequence similarity to the ATP-binding and presumed catalytic domain of the catalytic subunit of phosphorylase b kinase.
The 28-kDa calbindin-D is a major calcium-binding protein in the basilar papilla of the chick.
Authors: Oberholtzer J C , Buettger C , Summers M C , Matschinsky F M
Published: May 2006

In previous work we identified a basilar papilla protein (BPP23) that appears to be one of the most abundant soluble proteins in the basilar papilla of the chick cochlea. Here we report the purification of protein BPP23 from chick cochlea and the generation of a specific antiserum. Immunoblotting and immunoprecipitation experiments with this antiserum indicate that BPP23 is a calcium-binding protein very similar, if not identical, to avian calbindin, the 28-kDa vitamin D-dependent calcium-binding protein. Although the basilar papilla contains both receptor hair cells and supporting cells, immunocytochemical studies by others have localized calbindin-like immunoreactivity to the hair cells in the rat auditory receptor epithelium. Our estimates of the abundance of protein BPP23, assuming exclusive localization within the hair cell, indicate a concentration of at least 1 mM. Avian calbindin has four high-affinity (Kd = 0.5 X 10(-6)) calcium-binding sites. The presence of a specific calcium-binding activity at such high levels suggests an important function for cochlear calbindin (BPP23) in hair cell calcium homeostasis and auditory transduction.
Single-channel events and gating behavior of the cardiac gap junction channel.
Authors: Burt J M , Spray D C
Published: May 2006

The activity of gap junction channels between pairs of neonatal rat heart cells in culture was studied under control conditions and during uncoupling procedures by using dual whole-cell voltage clamp techniques. Under control conditions gap junctional conductance ranged from 0.05 to 35 nS. In cell pairs exhibiting low gap junctional conductance (less than 500 pS), single-channel events with a unitary conductance of 53 +/- 2 pS (5 experiments; 186 events) were apparent. Event duration and open-time probability were estimated to be 0.95 sec and 0.17, respectively. When the junctional conductance in well-coupled cell pairs (with initial junctional conductance, greater than 5 nS) was reduced by cytoplasmic acidification or application of heptanol, single-channel events could be visualized. Compared to low-conductance controls, unitary channel conductance was unaltered (for acidification the conductance was 58 +/- 3 pS in 11 experiments with 253 events; for heptanol the conductance was 61 +/- 1 pS in 2 experiments with 171 events), while the probability of channels being open was decreased. The constancy of unitary channel conductance under control conditions and during uncoupling procedures suggests that opening and closing of the gap junction channel are all-or-none processes during which no stable subconductance states are formed.
Developmental regulation of an insulin-degrading enzyme from Drosophila melanogaster.
Authors: Stoppelli M P , Garcia J V , Decker S J , Rosner M R
Published: May 2006

The precise mechanism by which insulin is degraded in mammalian cells is not presently known. Several lines of evidence suggest that degradation is initiated by a specific nonlysosomal insulin-degrading enzyme (IDE). The potential importance of this insulin protease is illustrated by the fact that there is an IDE in Drosophila melanogaster Kc cells that shares both physical and kinetic properties with its mammalian counterpart. We now demonstrate that the IDE is present in other Drosophila cell lines and in the embryo, the larvae, the pupae, and adult tissues of the fruit fly. Further, the level of the IDE is developmentally regulated, being barely detectable in the embryo but elevated approximately 5-fold in the larvae and pupae and approximately 10-fold in the adult fly. The IDE levels in the cell lines are particularly high, at least 10-fold greater than in the adult fly. Analysis of Schneider L3 cells indicates that the addition of the Drosophila hormone ecdysone, which induces differentiation of the cells, causes a small but reproducible increase in the level of the IDE and the insulin-degrading activity. These results demonstrate that the IDE is evolutionarily conserved and that its expression is tightly regulated during differentiation of Drosophila. The particular pattern of developmental regulation suggests that the IDE plays a specific and critical role in the later stages of the life cycle of the fly.
Spectrum of spontaneous mutation at the APRT locus of Chinese hamster ovary cells: an analysis at the DNA sequence level.
Authors: de Jong P J , Grosovsky A J , Glickman B W
Published: May 2006

The spectrum of spontaneous mutation of an endogenous mammalian cell gene has been determined at the DNA sequence level. Thirty independent spontaneous APRT- mutations were cloned and subsequently completely sequenced. Twenty-seven contained single base substitutions. Of these, 22 were G.C to A.T transitions, suggesting a major role for the deamination of cytosine in spontaneous mutagenesis of Chinese hamster ovary cells. The remaining mutants included a tandem double substitution, a -1 frameshift, and a 17-base-pair deletion flanked by a 2-base-pair direct repeat. Many of the independently recovered mutants were clustered at sites of multiple occurrence (hot spots). One site accounted for greater than 25% of all independently recovered events. Mutations were generally located within the coding sequence, although two mutations occurred within the consensus sequence for a 3' splice site.
Use of human peripheral blood lymphocytes to measure DNA binding capacity of chemical carcinogens.
Authors: Gupta R C , Earley K , Sharma S
Published: May 2006

Although animal models have been used successfully to study metabolic activation and binding of carcinogens to DNA, only limited studies have been done in human systems. To circumvent the problems associated with the inaccessibility of human tissues and a lack of sensitive methods to detect DNA damage, we have investigated the capability of human peripheral blood lymphocytes in vitro to metabolize carcinogens to their DNA binding species by a 32P-labeled adduct assay. Freshly isolated lymphocytes were exposed at 37 degrees C for 18 hr to 4-aminobiphenyl, 2-aminofluorene, 2-anthramine, 2-acetylaminophenanthrene, benzidine, 1-nitropyrene, 1,2-benzanthracene, triphenylene, 7,12-dimethylbenz[a]anthracene, or benzo[a]pyrene at 30 microM each, compounds that are shown or suspected to be carcinogenic in experimental animals. Anthracene, pyrene, and perylene were included as noncarcinogenic controls. Our data indicate that all test carcinogens formed readily measurable levels of DNA adducts. Analysis of exposed DNAs by 32P-labeling after digestion and adduct enrichment showed exclusively or predominantly one major adduct for all test carcinogens, except for 2-anthramine, triphenylene, and 7,12-dimethylbenz[a]anthracene, which showed two or three adducts, in the range of 8-1500 amol/micrograms of DNA. No DNA binding was detected for the noncarcinogens. From 12 lymphocyte specimens studied thus far, significant interindividual variations were observed for 2-aminofluorene (62-fold), 7,12-dimethylbenz[a]anthracene (10-fold), benzidine (19-fold) and benzo[a]pyrene (18-fold) in their capacity to bind to the lymphocyte DNA. The lymphocyte system in combination with the 32P-adduct assay may prove to be an ultrasensitive means to determine interindividual variations in the ability to biotransform carcinogens.
Molecular cloning of L-methylmalonyl-CoA mutase: gene transfer and analysis of mut cell lines.
Authors: Ledley F D , Lumetta M , Nguyen P N , Kolhouse J F , Allen R H
Published: May 2006

L-Methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) is a mitochondrial adenosylcobalamin-requiring enzyme that catalyzes the isomerization of L-methylmalonyl-CoA to succinyl-CoA. This enzyme is deficient in methylmalonic acidemia, an often fatal disorder of organic acid metabolism. Antibody against human placental MCM was used to screen human placenta and liver cDNA expression libraries for MCM cDNA clones. One clone expressed epitopes that could affinity-purify antibodies against MCM. A cDNA corresponding in length to the mRNA was obtained and introduced into COS cells by DNA-mediated gene transfer. Cells transformed with this clone expressed increased levels of MCM enzymatic activity. RNA blot analysis of cells genetically deficient in MCM indicates that several deficient cell lines have a specific decrease in the amount of hybridizable mRNA. These data confirm the authenticity of the MCM cDNA clone, establish the feasibility of constituting MCM activity by gene transfer for biochemical analysis and gene therapy, and provide a preliminary picture of the genotypic spectrum underlying MCM deficiency.
The mouse surfeit locus contains a very tight cluster of four "housekeeping" genes that is conserved through evolution.
Authors: Williams T , Yon J , Huxley C , Fried M
Published: May 2006

The four identified alternating transcription units (Surf-1 to Surf-4) in the mouse surfeit locus are very tightly clustered, no two neighboring units being separated by more than 73 base pairs and the Surf-2 and Surf-4 transcription units overlapping by 133 base pairs at their 3' ends. All four surfeit genes, which are unrelated by sequence similarity, were found to have the properties of "housekeeping" genes, being expressed in a variety of differentiated mouse cell lines and containing unmethylated CpG-rich islands in the vicinity of their 5' ends. The unusual organization of the four surfeit genes was found not to be unique to the mouse: the same juxtaposition of the genes was found to be conserved in a number of different mammals, including humans. The four human surfeit genes were also found to be transcriptionally active.
Evidence that DNA is present in abnormal tubulofilamentous structures found in scrapie.
Authors: Narang H K , Asher D M , Gajdusek D C
Published: May 2006

Abnormal tubulofilamentous structures have been identified in electron micrographs of thin sections and negatively stained impression grids prepared from brains of animals with scrapie and other spongiform encephalopathies, and we showed that such tubules contain a core of filamentous structures resembling scrapie-associated fibrils (SAF). We treated impression grids from brains of scrapie-infected hamsters with several substances that bind to or cleave proteins and nucleic acids to see if they had any effect on the abnormal tubulofilamentous structures. Treatment with three proteolytic enzymes reduced the caliber of the tubules from about 50 nm to 30 nm; subsequent treatment of the 30-nm tubules with DNase I left many typical SAF as well as transitional forms in which twisted SAF emerged from tubules. DNase treatment of the original thicker tubules had no effect, and no SAF were seen on grids. Treatment of the 30-nm tubules with any of three other nucleases (micrococcal, mung bean, and BAL-31) also produced SAF. However, treatment with RNase A had no effect either on the original 50-nm tubules or on the 30-nm tubules produced by proteolysis. Detergent treatment of any of the preparations produced SAF. Treatment with ethidium bromide resulted in staining of the tubules that was inhibited by magnesium ions. The data suggest that the abnormal tubulofilamentous particles found in spongiform encephalopathies may consist of an outer cylinder of protein, an inner cylinder of DNA, and an innermost core of SAF.
Transgenic mice express the human phenylethanolamine N-methyltransferase gene in adrenal medulla and retina.
Authors: Baetge E E , Behringer R R , Messing A , Brinster R L , Palmiter R D
Published: May 2006

The human gene for phenylethanolamine N-methyltransferase (hPNMT), responsible for the conversion of norepinephrine to epinephrine, has been cloned and the complete nucleotide sequence has been determined. The structural gene consists of three exons and two introns spanning approximately equal to 2100 base pairs. Transgenic mice containing the hPNMT gene with either 2 or 8 kilobases of 5'-flanking sequence were produced and resulted in expression of hPNMT mRNA in the adrenal gland and eye. A chimeric gene consisting of 2 kilobases of the hPNMT 5'-flanking region fused to the simian virus 40 early region also resulted in tumor (T) antigen mRNA expression in adrenal glands and eyes; furthermore, immunocytochemistry showed that tumor antigen was localized in nuclei of adrenal medullary cells and cells of the inner nuclear cell layer of the retina, prominent sites of epinephrine synthesis. These results indicate that the enhancer(s) for appropriate expression of the hPNMT gene in these cell types is in the 2-kilobase 5'-flanking region of the human gene.
Factor inducing
Agrobacterium tumefaciens vir
gene expression is present in monocotyledonous plants
Authors: Usami Shoji , Okamoto Shigehisa , Takebe Itaru , Machida Yasunori
Published: May 2006

Agrobacterium tumefaciens
harboring the tumor-inducing Ti plasmid incites crown gall tumor on dicotyledonous species. Upon infection of these plants, Ti plasmid DNA sequence is stably transferred (T-DNA) by unknown mechanisms to plant cells to be integrated into nuclear DNA. The T-DNA processing and transfer require the expression of
vir
(virulence) genes on the Ti plasmid, which are known to be induced by certain phenolic compounds released from cells at the wounded inoculation site. The results of the present study demonstrate that wheat and oats contain a substance(s) that induces
vir
gene expression, yet the inducing substance of wheat differs from the phenolic inducers in that it is hydrophilic and has a molecular weight of several thousand. The novel inducer was not detectable in the exudates of seedlings of these plants but was found in an extract from the transition region between shoot and root of the seedlings and also in extracts from the seeds, bran, and germ. This finding suggests that T-DNA processing and possibly its transfer should take place when
Agrobacterium
invades suitable tissues of monocotyledonous plants.
On the evolution of the adaptation of
Lophopyrum elongatum
to growth in saline environments
Authors: Dvořák Jan , Edge Mark , Ross Kathleen
Published: May 2006

Most species of the genus
Lophopyrum
Löve (
Agropyron
Geartn.) grow in saline environments and are more tolerant of saline stress than the species of the related genus
Triticum
L. A 56-chromosome amphiploid from the cross
Triticum aestivum
cv. Chinese Spring ×
Lophopyrum elongatum
exceeded Chinese Spring in salt tolerance, measured as plant dry-matter production and seed yield in solution cultures with 250 mM NaCl. Thus, the adaptation of
Lophopyrum
to saline environments is expressed in the wheat genetic background. None of the disomic additions or substitutions of
L. elongatum
chromosomes in Chinese Spring showed a similar level of saline stress tolerance, which indicates that the trait depends on the activity of genes on more than one chromosome. Comparisons of disomic additions, double monosomic additions from half-diallel crosses among disomic additions, and disomic substitutions of
L. elongatum
chromosomes in Chinese Spring with Chinese Spring indicated that the enhanced salt tolerance of the amphiploid is primarily controlled by genes with minor effects on three of the seven chromosomes, 3E, 4E, and 7E, interacting in a largely additive manner. The salt tolerance of
L. elongatum
additionally depends on several minor nonadditive gene interactions. It is concluded that the adaptation of
L. elongatum
to growth in saline environments evolved by accumulation of new alleles in a number of loci, each with a relatively small effect on salt tolerance. It is further inferred that most of these new alleles were codominant to the original alleles and were able to act independently in enhancing salt tolerance.
A liver-specific DNA-binding protein recognizes multiple nucleotide sites in regulatory regions of transthyretin, alpha 1-antitrypsin, albumin, and simian virus 40 genes.
Authors: Costa R H , Grayson D R , Xanthopoulos K G , Darnell J E
Published: May 2006

Double-stranded oligodeoxynucleotides that represent protein binding sites in the regulatory regions of the mouse genes encoding transthyretin (TTR) and alpha 1-antitrypsin (alpha 1-AT) bound a nuclear protein factor(s) found mainly in hepatocytes. A site in the regulatory region of the gene encoding rat serum albumin and, surprisingly, a region in the simian virus 40 enhancer also bind the same factor. Oligodeoxynucleotide affinity chromatography (with one of the TTR binding sites) allowed a 500-fold purification of the protein. The purified protein protected similar portions of all the regulatory regions, as well as the simian virus 40 core C enhancer element, from digestion with DNase I. A DNA-binding protein previously purified from liver by virtue of its ability to bind to several virus enhancer sequences also binds to TTR, alpha 1-AT, and albumin regulatory sites. Thus, all these binding sites, which contain only minimal sequence similarity, may bind to a single protein, or a similar family of proteins, that activates liver-specific transcription of coordinately expressed genes.
Exposure of salivary gland cells to low-frequency electromagnetic fields alters polypeptide synthesis.
Authors: Goodman R , Henderson A S
Published: May 2006

This study demonstrates that exposure of cells to extremely low-frequency electromagnetic fields can cause measurable changes in protein synthesis. Sciara coprophila salivary gland cells were exposed to five low-frequency (1.5-72 Hz) electromagnetic signals: three signals (1.5, 15, and 72 Hz) produced pulsed asymmetric electromagnetic fields and two signals (60 and 72 Hz) were sinusoidal. Subsequent analyses of two-dimensional gels showed that cell exposure to either type of low-frequency electromagnetic field resulted in both qualitative and quantitative changes in patterns of protein synthesis. Thus, signals producing diverse waveform characteristics induced previously undetectable polypeptides, some of which were signal specific and augmented or suppressed other polypeptides as compared with nonexposed cells. The pattern of polypeptide synthesis differed from that seen with heat shock: only five polypeptides in cells exposed to electromagnetic signals overlap those polypeptides exposed to heat shock, and the suppression of protein synthesis characteristic of heat shock does not occur.
Photophobe (Ppb), a Drosophila mutant with a reversed sign of phototaxis; the mutation shows an allele-specific interaction with sevenless.
Authors: Ballinger D G , Benzer S
Published: May 2006

We have isolated a dominant behavioral mutation, Photophobe (Ppb), on the second chromosome of Drosophila melanogaster. Although wild-type flies are attracted towards green light, flies homozygous for the Ppb mutation avoid it over an intensity range of six logarithms. Ppb interacts in a dominant way with mutations in the sevenless (sev) gene, an X-chromosomal gene necessary for photoreceptor cell 7 differentiation in the Drosophila retina. Specific alleles of sev alter the Ppb behavioral phenotype; of eight sev alleles tested, two alleles enhanced the negative phototaxis of Ppb, whereas six alleles had the opposite effect. In no mutant combination of Ppb and sev was photoreceptor cell 7 restored. These data show that the sev gene, in addition to its role in the differentiation of photoreceptor cell 7, plays a role along with Ppb in a common visual information-processing pathway.
Nature of polymorphism in HLA-A, -B, and -C molecules.
Authors: Parham P , Lomen C E , Lawlor D A , Ways J P , Holmes N , Coppin H L , Salter R D , Wan A M , Ennis P D
Published: May 2006

Diversity in 39 HLA-A, -B, and -C molecules is derived from 20 amino acid positions of high variability and 71 positions of low variability. Variation in the structurally homologous alpha 1 and alpha 2 domains is distinct and may correlate with partial segregation of peptide and T-cell receptor binding functions. Comparison of 15 HLA-A with 20 HLA-B molecules reveals considerable locus-specific character, due primarily to differences at polymorphic residues. The results indicate that genetic exchange between alleles of the same locus has been a more important mechanism in the generation of HLA-A, -B, and -C diversity than genetic exchange events between alleles of different loci.
Altered regulation of lipid biosynthesis in a mutant of
Arabidopsis
deficient in chloroplast glycerol-3-phosphate acyltransferase activity
Authors: Kunst Ljerka , Browse John , Somerville Chris
Published: May 2006

The leaf membrane lipids of many plant species, including
Arabidopsis thaliana
(L.) Heynh., are synthesized by two complementary pathways that are associated with the chloroplast and the endoplasmic reticulum. By screening directly for alterations in lipid acyl-group composition, we have identified several mutants of
Arabidopsis
that lack the plastid pathway because of a deficiency in activity of the first enzyme in the plastid pathway of glycerolipid synthesis, acyl-ACP:
sn
-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) (where ACP is acyl carrier protein). The lesion results in an increased synthesis of lipids by the cytoplasmic pathway that largely compensates for the loss of the plastid pathway and provides nearly normal amounts of all the lipids required for chloroplast biogenesis. However, the fatty acid composition of the leaf membrane lipids of the mutants is altered because the acyltransferases associated with the two pathways normally exhibit different substrate specificities. The remarkable flexibility of the system provides an insight into the nature of the regulatory mechanisms that allocate lipids for membrane biogenesis.
Mutant 16S ribosomal RNA: a codon-specific translational suppressor.
Authors: Murgola E J , Hijazi K A , Göringer H U , Dahlberg A E
Published: May 2006

We have isolated an unusual codon-specific translational suppressor in Escherichia coli. The suppressor resulted from a spontaneous mutation in a chromosomal gene during a selection for suppressors of the auxotrophic nonsense mutation trpA(UGA211). The suppressor allows readthrough of UGA mutations at two positions in trpA and at two sites in bacteriophage T4. It does not, however, suppress amber (UAG) or ochre (UAA) mutations that were tested in both genomes, some of which were at the same positions as the suppressible UGA mutations. The suppressor also does not allow mistranslation of the UGA-related trpA missense mutations UGG at positions 211 and 234, AGA at 211 and 234, CGA at 211, or UGU and UGC at 234. The suppressor mutation was mapped by genetic procedures to position 89 on the E. coli genetic map. Localization of the suppressor mutation to rrnB was achieved by cloning it in the low-copy-number plasmid pEJM007 by in vivo recombination from the chromosome. Recloning in bacteriophage M13 and subsequent DNA sequence analysis allowed the identification of the suppressor mutation as a deletion of the cytidylic acid residue at nucleotide position 1054 of the 16S ribosomal RNA. The mutant EcoRI-Xba I fragment from the suppressor gene was recloned, from M13, in an otherwise wild-type rrnB in the plasmid pEJM007, and UGA suppression was examined. The UGA-suppressing activity of the reconstructed suppressor-containing pEJM007 was indistinguishable from that of the original recombinant suppressor-containing plasmid. This result demonstrates that the C1054 deletion in 16S rRNA is both necessary and sufficient for UGA suppression. The existence of this mutant suggests an important role for rRNA in codon recognition, at least for accurate polypeptide chain termination.
cDNA cloning, sequence, and regulation of a major female-specific and growth hormone-inducible rat liver cytochrome P-450 active in 15 beta-hydroxylation of steroid sulfates.
Authors: Zaphiropoulos P G , Mode A , Ström A , Möller C , Fernandez C , Gustafsson J A
Published: May 2006

cDNA clones encompassing the complete coding sequence for the female-specific rat liver microsomal 15 beta-hydroxylase have been isolated and sequenced. This cytochrome P-450 species (P-450 15 beta) has a total of 490 amino acid residues, and its deduced amino acid sequence as well as direct sequencing of the amino-terminal portion of the purified protein revealed its identity with P-450 i. P-450 15 beta was found to be 66% similar to the male-specific rat liver microsomal 16 alpha-hydroxylase, P-450 16 alpha. Furthermore, P-450 15 beta is 47% similar to the major phenobarbital-inducible cytochrome P-450 in rat liver, P-450 b, while its structural similarity to P-450 c, P-450 pregnenolone-16a-carbonitrile, and P-450 lauric acid omega-hydroxylase is less than 30%. However, the developmentally regulated cytochromes P-450 f and P-450 PB-1 both have 68% similarity to P-450 15 beta. It is therefore concluded that P-450 15 beta belongs to the same gene subfamily (P450IIC) that also includes cytochromes P-450 f, PB-1, and 16 alpha. The plasma pattern of growth hormone (GH) is known to be a major determinant for the sex-specific expression of P-450 15 beta and P-450 16 alpha. Continuous infusion of GH to hypophysectomized male rats causes an increase in P-450 15 beta mRNA after 6 days of treatment, while intermittent injections have no effect. On the other hand, no effect on P-450 16 alpha mRNA is observed after continuous administration of GH, while intermittent injections cause an increase after 2 days of treatment. This relatively long period before P-450 15 beta and P-450 16 alpha mRNA induction is seen might indicate that protein factors mediating GH action are involved.
Small ribonucleoproteins in Schizosaccharomyces pombe and Yarrowia lipolytica homologous to signal recognition particle.
Authors: Poritz M A , Siegel V , Hansen W , Walter P
Published: May 2006

We have partially purified ribonucleoproteins (RNPs) from Schizosaccharomyces pombe and Yarrowia lipolytica with properties resembling those of mammalian signal recognition particle (SRP). In both species of yeast we have identified a single major RNA species in the size range of SRP RNA (256 nucleotides in S. pombe and 270 nucleotides in Y. lipolytica) present in postribosomal salt extracts of the cytoplasm. The RNPs containing these RNAs sediment in sucrose gradients at 11 S and 10 S for S. pombe and Y. lipolytica, respectively. Analysis of genomic clones of these RNAs has revealed that (i) they are encoded by single copy genes; (ii) they share two short conserved sequences that match the A and B boxes defined for polymerase III promoters; (iii) they can be folded into secondary structures that closely match that defined by phylogenetic analysis of higher eukaryotic SRP RNAs; and (iv) they show primary sequence conservation in short regions predicted to be single stranded. Both of the yeast RNAs bind under stringent conditions to canine SRP proteins. Most importantly, RNase protection of the S. pombe RNA by the individual canine SRP proteins, p19 and p68/72, shows that the proteins recognize homologous elements of the mammalian and yeast RNA. Taken together these data suggest strongly that we have identified yeast SRP homologues.
Negative regulation of DNA synthesis in early erythropoietic progenitor cells (BFU-E) by a protein purified from the medium of C57BL/6 mouse marrow cells.
Authors: Del Rizzo D F , Eskinazi D , Axelrad A A
Published: May 2006

During studies on the influence of Fv-2 on the cycle state of the erythroid burst-forming unit (BFU-E), an activity was found in bone marrow supernatants from C57BL/6 (B6) mice that shut down DNA synthesis of the BFU-E in vitro. It acted within minutes, its action was completely reversed by a single wash, and it appeared specific to the BFU-E. The activity-causing substance, being macromolecular, heat stable, and trypsin-sensitive, was evidently a protein and was named negative regulatory protein. We purified a negative regulatory protein from a bone marrow-derived "B6 Pan" cell line with properties thus far indistinguishable from those of the negative regulatory protein obtained directly from B6 marrow. By gel filtration the protein has a Mr of approximately equal to 79,000, by cation- and anion-exchange chromatography it appears to be a neutral molecule at physiological pH, and the molecule does not bind to Con A. After five sequential chromatographic steps, we obtained a preparation active at a concentration of 25 ng/ml. Our findings are compatible with the hypothesis that quiescence of BFU-E with respect to DNA synthesis in vivo in B6 mice is mediated by negative regulatory protein molecules.
Heterotypic and homotypic associations between the nuclear lamins: site-specificity and control by phosphorylation.
Authors: Georgatos S D , Stournaras C , Blobel G
Published: May 2006

Using purified components in affinity chromatography and blot binding assays, we have found that rat liver lamins A, B, and C can associate in homotypic and heterotypic fashions. Heterotypic A-B and C-B complexes are unusually stable and involve the common amino-terminal domain of lamins A and C, but not their helical "rod" domain. A synthetic peptide, comprising the first 32 amino acid residues of lamins A and C, is able to fully compete with the intact molecules for binding to lamin B. Conversely, heterotypic A-C associations and homotypic A-A and C-C interactions appear significantly weaker than A/C-B binding and do not involve the lamin A and C amino-terminal domain. Homotypic B-B complexes are not formed to any considerable extent unless isolated lamin B subunits are "superphosphorylated" in vitro with protein kinase A. However, when lamins A and C are similarly modified, no changes in their binding specificity can be detected. These data suggest that the nuclear lamina, unlike other multicomponent intermediate filaments, constitutes a nonobligatory heteropolymer. They also indicate that cAMP-dependent phosphorylation of interphase lamin B could cause remodeling of the lamina and establishment of homopolymeric domains.
Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations.
Authors: Cotton R G , Rodrigues N R , Campbell R D
Published: May 2006

The chemical reactivity of thymine (T), when mismatched with the bases cytosine, guanine, and thymine, and of cytosine (C), when mismatched with thymine, adenine, and cytosine, has been examined. Heteroduplex DNAs containing such mismatched base pairs were first incubated with osmium tetroxide (for T and C mismatches) or hydroxylamine (for C mismatches) and then incubated with piperidine to cleave the DNA at the modified mismatched base. This cleavage was studied with an internally labeled strand containing the mismatched T or C, such that DNA cleavage and thus reactivity could be detected by gel electrophoresis. Cleavage at a total of 13 T and 21 C mismatches isolated (by at least three properly paired bases on both sides) single-base-pair mismatches was identified. All T or C mismatches studied were cleaved. By using end-labeled DNA probes containing T or C single-base-pair mismatches and conditions for limited cleavage, we were able to show that cleavage was at the base predicted by sequence analysis and that mismatches in a length of DNA could be readily detected by such an approach. This procedure may enable detection of all single-base-pair mismatches by use of sense and antisense probes and thus may be used to identify the mutated base and its position in a heteroduplex.
DNA base sequence changes and sequence specificity of bromodeoxyuridine-induced mutations in mammalian cells.
Authors: Davidson R L , Broeker P , Ashman C R
Published: May 2006

By using a shuttle vector system developed in our laboratory, we have carried out studies on the molecular mechanism by which 5-bromodeoxyuridine (BrdUrd) induces mutations in mammalian cells. The target for mutagenesis in these studies was the Escherichia coli gpt gene that was contained within a retroviral shuttle vector and integrated into chromosomal DNA in mouse A9 cells. Shuttle vector-transformed cells expressing the gpt gene were mutagenized with BrdUrd and cells with mutations in the gpt gene were selected. Shuttle vector sequences were recovered from the mutant cells, and the base sequence of the mutant gpt genes was determined. The great majority of the BrdUrd-induced mutations involving single-base changes were found to be G.C----A.T transitions. We have shown that mutagenesis by BrdUrd depends upon perturbation of deoxycytidine metabolism. Thus, the current results suggest that BrdUrd mutagenesis involves mispairing and misincorporation of BrdUrd opposite guanine in DNA, driven by nucleotide pool perturbation caused by BrdUrd and the resulting imbalanced supply of triphosphates available for DNA synthesis. The results also revealed a very high degree of sequence specificity for the BrdUrd mutagenesis. BrdUrd-induced G.C----A.T transitions occurred almost exclusively in sequences with two adjacent guanine residues. Furthermore, in approximately equal to 90% of the cases, the guanine residue involved in mutation was the one in the more 3' position.
Characterization of frequent deletions causing steroid 21-hydroxylase deficiency.
Authors: White P C , Vitek A , Dupont B , New M I
Published: May 2006

Steroid 21-hydroxylase deficiency is caused by mutations in the CYP21B gene. This gene and a highly homologous pseudogene, CYP21A, alternate with the C4A and C4B genes encoding the fourth component of complement. Classical deficiency alleles are frequently caused by deletions of CYP21B or by gene conversions that transfer deleterious mutations from the CYP21A pseudogene to CYP21B. Gene conversions involving restriction enzyme sites that distinguish CYP21A [e.g., 3.2-kilobase (kb) Taq I fragment] and CYP21B (3.7-kb Taq I fragment) might be confused with actual deletions of CYP21B. To determine the incidence of this type of gene conversion, 15 chromosomes (in 13 families) with absent 3.7-kb Taq I fragments were studied. When hybridized with a 21-hydroxylase probe, all of these chromosomes were associated with absent 2.9-kb Kpn I fragments, 14 of 15 were associated with absent 2.4-kb Bgl II/EcoRI fragments, and 13 of 15 were associated with absent 10-kb Bgl II/EcoRI and 12-kb EcoRI fragments. Thirteen of 15 chromsomes had absent 6.0- or 5.4-kb Taq I fragments when hybridized with a C4 probe. Thus, 2 of 15 chromosomes do not carry deletions and may represent gene conversions; 13 of 15 chromosomes studied have a deletion of approximately equal to 30 kb, leaving behind the C4A gene and a single CYP21A-like gene. Hybridization with specific oligonucleotide probes showed that in all 13 cases this remaining CYP21 gene carried an 8-base-pair deletion, typical of CYP21A, that prevents synthesis of a functional protein. Thus, gene conversions are rarely confused with deletions as a cause of 21-hydroxylase deficiency.
Elimination of the adverse effects of urea fertilizer on seed germination, seedling growth, and early plant growth in soil
Authors: Bremner John M. , Krogmeier Michael J.
Published: May 2006

The rapidly increasing importance of urea fertilizer in world agriculture has stimulated research to find methods of reducing the problems associated with the use of this fertilizer. One of these problems is that urea has adverse effects on seed germination, seedling growth, and early plant growth in soil. Because there is evidence that these adverse effects are caused largely, if not entirely, by ammonia produced through hydrolysis of urea fertilizer by soil urease, we explored the possibility that they could be reduced or eliminated by amending urea fertilizer with a small amount of a urease inhibitor. Studies with seeds of alfalfa (
Medicago sativa
L.), barley (
Hordeum vulgare
L.), oats (
Avena sativa
L.), rye (
Secale cereale
L.), sorghum [
Sorghum bicolor
(L.) Moench], and wheat (
Triticum aestivum
L.) showed that phenylphosphorodiamidate and
N
-(
n
-butyl)thiophosphoric triamide were the most effective of 10 urease inhibitors evaluated for reduction of the adverse effect of urea on seed germination.
N
-(
n
-butyl)-thiophosphoric triamide was superior to phenylphosphorodiamidate for reducing the adverse effects of urea solutions on seed germination and seedling growth in soil, and it completely eliminated the adverse effect of urea granules on early plant growth in soil. The data reported indicate that the adverse effects of urea fertilizer on seed germination, seedling growth, and early plant growth in soil could be eliminated or markedly reduced by amending the fertilizer with as little as 0.01% (wt/wt) of
N
-(
n
-butyl)thiophosphoric triamide.
Two mouse genes encoding potential transcription factors with identical DNA-binding domains are activated by growth factors in cultured cells.
Authors: Lemaire P , Revelant O , Bravo R , Charnay P
Published: May 2006

We previously reported the identification of a mouse gene, Krox-20, encoding a protein with three "zinc fingers" (DNA-binding domains with coordinated zinc ions) whose expression is regulated during G0/G1 transition (cell-cycle reentry). We now have isolated cDNAs corresponding to a related gene, Krox-24. Krox-24 encodes a protein with zinc fingers nearly identical to those encoded by Krox-20 and similar to those of transcription factor Sp1. Similarity between Krox-20 and Krox-24 proteins also extends to several blocks of amino acid sequence located upstream of the finger region. Like Krox-20, Krox-24 is transiently activated in quiescent cells after treatment with fetal bovine serum or purified growth factors. The kinetics of activation are similar to those of the protooncogene c-fos. The induction does not require de novo protein synthesis, and cycloheximide treatment of the cells leads to superinduction due, at least in part, to mRNA stabilization. In the mouse, the two genes are expressed in a tissue-specific manner, with slightly different patterns. These properties suggest that Krox-20 and Krox-24 may encode transcription factors with identical DNA target sequences and that these factors may be involved in the modulation of cell proliferation and differentiation.
Molecular cloning of the common acute lymphoblastic leukemia antigen (CALLA) identifies a type II integral membrane protein.
Authors: Shipp M A , Richardson N E , Sayre P H , Brown N R , Masteller E L , Clayton L K , Ritz J , Reinherz E L
Published: May 2006

Common acute lymphoblastic leukemia antigen (CALLA) is a 100-kDa cell-surface glycoprotein expressed on most acute lymphoblastic leukemias and certain other immature lymphoid malignancies and on normal lymphoid progenitors. The latter are either uncommitted to B- or T-cell lineage or committed to only the earliest stages of B- or T-lymphocyte maturation. To elucidate to homogeneity, obtained the NH2-terminal sequence from both the intact protein and derived tryptic and V8 protease peptides and isolated CALLA cDNAs from a Nalm-6 cell line lambda gt10 library using redundant oligonucleotide probes. The CALLA cDNA sequence predicts a 750-amino acid integral membrane protein with a single 24-amino acid hydrophobic segment that could function as both a transmembrane region and a signal peptide. The COOH-terminal 700 amino acids, including six potential N-linked glycosylation sites compose the extracellular protein segment, whereas the 25 NH2-terminal amino acids remaining after cleavage of the initiation methionine form the cytoplasmic tail. CALLA+ cells contain CALLA transcripts of 2.7 to 5.7 kilobases with the major 5.7- and 3.7-kilobase mRNAs being preferentially expressed in specific cell types.
Structure and synthesis of a potent glutamate receptor antagonist in wasp venom.
Authors: Eldefrawi A T , Eldefrawi M E , Konno K , Mansour N A , Nakanishi K , Oltz E , Usherwood P N
Published: May 2006

A low molecular weight toxin isolated from the venom of the digger wasp Philanthus triangulum, first noted by T. Piek, is a potent antagonist of transmission at quisqualate-sensitive glutamate synapses of locust leg muscle. This philanthotoxin 433 (PTX-433) has been purified, chemically characterized, and subsequently synthesized along with two closely related analogues. It has a butyryl/tyrosyl/spermine sequence and a molecular weight of 435. Its two analogues, PTX-343 and PTX-334 (the numerals denoting the number of methylenes between the amino groups of the spermine moiety), are also active on the glutamate synapse of the locust leg muscle; PTX-334 was more potent and PTX-343 was less potent than the natural toxin. Such chemicals are useful for studying, labeling, and purifying glutamate receptors and may become models for an additional class of therapeutic drugs and possibly insecticides.
Molecular model for receptor-stimulated calcium spiking.
Authors: Meyer T , Stryer L
Published: May 2006

Many cells exhibit periodic transient increases in cytosolic calcium levels rather than a sustained rise when stimulated by a hormone or growth factor. We propose here a molecular model that accounts for periodic calcium spiking induced by a constant stimulus. Four elements give rise to repetitive calcium transients: cooperativity and positive feedback between a pair of reciprocally coupled (crosscoupled) messengers, followed by deactivation and then by reactivation. The crosscoupled messengers in our model are inositol 1,4,5-trisphosphate (InsP3) and cytosolic calcium ions. The opening of calcium channels in the endoplasmic reticulum by the binding of multiple molecules of InsP3 provides the required cooperativity. The stimulation of receptor-activated phospholipase C by released calcium ions leads to positive feedback. InsP3 is destroyed by a phosphatase, and calcium ion is pumped back into the endoplasmic reticulum. These processes generate bistability: the cytosolic calcium concentration abruptly increases from a basal level to a stimulated level at a threshold degree of activation of phospholipase C. Spiking further requires slow deactivation and subsequent reactivation. In our model, mitochondrial sequestration of calcium ion prevents the cytosolic level from increasing above several micromolar and enables the system to return to the basal state. When the endoplasmic reticulum calcium store is refilled to a critical level by the Ca2+-ATPase pump, cooperative positive feedback between the InsP3-gated channel and phospholipase C begins again to give the next calcium spike. The time required for the calcium level in the endoplasmic reticulum to reach a threshold sets the interval between spikes. The amplitude, shape, and period of calcium spikes calculated for this model are like those observed experimentally.