bluuebunny/crossref_metadata_2025 huggingface dataset:
Linkage map of three HLA-DR beta-chain genes: evidence for a recent duplication event.
Authors: Rollini P , Mach B , Gorski J
Published: May 2006

The predominant class II, or Ia, antigen of the human major histocompatibility complex is HLA-DR. It consists of an alpha and a beta chain, the latter being responsible for the remarkable polymorphism of these Ia antigens. Studies with cloned genes had shown the existence of more than one DR beta-chain locus. We have isolated about 100 kilobases of the HLA-DR beta-chain gene region from a cosmid library generated from a consanguineous homozygous B-cell line of the DR3 haplotype. Three HLA-DR beta-chain genes have been characterized. They are arranged in a head-to-tail orientation. One of the genes lacks the region encoding the first domain of the DR beta chain. The two other genes are transcribed, as shown by RNA blot hybridization analysis. A striking restriction site homology has been found within the DR beta-chain gene cluster, suggesting a recent duplication event involving at least 25 kilobases of DNA. Moreover, the molecular map of DR beta chain genes cloned from B-cell lines of two other HLA-DR haplotypes shows extensive homology between alleles of a given DR beta-chain locus.
Activated and unactivated forms of human erythrocyte aldose reductase.
Authors: Srivastava S K , Hair G A , Das B
Published: May 2006

Aldose reductase (alditol:NADP+ 1-oxidoreductase, EC 1.1.1.21) has been partially purified from human erythrocytes by DEAE-cellulose (DE-52) column chromatography. This enzyme is activated severalfold upon incubation with 10 microM each glucose 6-phosphate, NADPH, and glucose. The activation of the enzyme was confirmed by following the oxidation of NADPH as well as the formation of sorbitol with glucose as substrate. The activated form of aldose reductase exhibited monophasic kinetics with both glyceraldehyde and glucose (Km of glucose = 0.68 mM and Km of glyceraldehyde = 0.096 mM), whereas the native (unactivated) enzyme exhibited biphasic kinetics (Km of glucose = 9.0 and 0.9 mM and Km of glyceraldehyde = 1.1 and 0.14 mM). The unactivated enzyme was strongly inhibited by aldose reductase inhibitors such as sorbinil, alrestatin, and quercetrin, and by phosphorylated intermediates such as ADP, glycerate 3-phosphate, glycerate 1,3-bisphosphate, and glycerate 2,3-trisphosphate. The activated form of the enzyme was less susceptible to inhibition by aldose reductase inhibitors and phosphorylated intermediates.
Deduced product of the stage 0 sporulation gene spo0F shares homology with the Spo0A, OmpR, and SfrA proteins.
Authors: Trach K A , Chapman J W , Piggot P J , Hoch J A
Published: May 2006

The location of the stage 0 sporulation locus spo0F has been determined on a cloned fragment of Bacillus subtilis DNA. The spo0F gene and surrounding region was sequenced and was shown to code for a protein of Mr 14,286. The amino acid sequence of this deduced protein was 56% homologous to the amino-terminal domain of the spo0A gene product. The molecular weight of the Spo0F protein was approximately half that of the Spo0A protein, and its sequence was homologous to the amino-terminal half of the Spo0A protein. This same portion of the Spo0A protein showed ancestral relationship to the OmpR and SfrA regulatory proteins of Escherichia coli. Mutations in any of the genes encoding these proteins in either organism are highly pleiotropic and result in alterations in the regulation of membrane components, suggesting that they may have related roles in both organisms and that the stage 0 sporulation defect of spo0A and spo0F mutants is an indirect consequence of this regulatory system.
Evidence that intracellular magnesium is present in cells at a regulatory concentration for protein synthesis.
Authors: Terasaki M , Rubin H
Published: May 2006

When extracellular magnesium is reduced by a factor of 50 (from 1.0 to 0.02 mM), the total intracellular magnesium of a spontaneously transformed clone of 3T3 cells decreases by 30-50%. Protein synthesis rates in these cells were measured as the intracellular magnesium decreased. Protein synthesis rates and magnesium content were found to decrease in parallel with each other. At 3 hr, a decrease to 84% of control values of magnesium content was accompanied by a decrease to 85% of control values of leucine incorporation rates. A larger inhibition had occurred by 12 hr, when the magnesium had decreased to 67% and leucine incorporation rates had decreased to 57%. When magnesium was restored to magnesium-deprived cells, both magnesium content and leucine incorporation increased about 2-fold by 1 hr. In the experiments reported here, initial small changes in magnesium content are associated with changes in protein synthesis rates. This strongly suggests that magnesium is present at a regulatory rather than excess concentration for protein synthesis. The results are consistent with a role for intracellular magnesium in the regulation of protein synthesis and support the hypothesis that magnesium has a central role in the regulation of metabolism and growth.
Molecular analysis of the yellow gene (y) region of Drosophila melanogaster.
Authors: Biessmann H
Published: May 2006

The yellow gene (y) is involved in pattern-specific melanin pigmentation of the cuticle of the adult fly and of larval mouth parts of Drosophila melanogaster. I have isolated some 70 kilobases (kb) of contiguous DNA from the y region. Chromosomal aberrations of y1-type alleles (null alleles) and y2-type alleles that give rise to characteristic pattern mosaicism of pigmentation were mapped by Southern blot analysis. The y2 allele is associated with the insertion of a "gypsy" transposable element 0.9 kb distal to the putative y coding region. A 3.1-kb region to which breakpoints of all y1-type alleles could be mapped is homologous to a 2.0-kb polyadenylylated mRNA, the expression of which is specifically regulated in development. This putative y gene transcript is present at high levels in pupae when melanization of the adult cuticle occurs, but its steady-state levels change dramatically during development, being highest in late embryos prior to hatching. This suggests that, in addition to melanin synthesis and/or deposition, the y gene product may have a role in other possibly neural functions.
Monoclonal antibody that defines human myoepithelium.
Authors: Dairkee S H , Blayney C , Smith H S , Hackett A J
Published: May 2006

We have isolated a mouse monoclonal antibody that, upon immunohistochemical localization in frozen sections, displays specificity for human myoepithelial cells in the resting mammary gland, sweat glands, and salivary glands. Furthermore, this antibody was strongly and homogeneously reactive with frozen sections of 3 of 60 breast carcinoma specimens. Using immunolocalization techniques in conjunction with polyacrylamide gel electrophoresis, we have determined that the reactivity of this monoclonal antibody is directed toward a 51,000-dalton keratin polypeptide. The potential uses of this antibody in the prognosis of human mammary carcinoma and in understanding the role of the myoepithelium in development and differentiation are discussed.
A defined system for the DNA strand-transfer reaction at the initiation of bacteriophage Mu transposition: protein and DNA substrate requirements.
Authors: Craigie R , Arndt-Jovin D J , Mizuuchi K
Published: May 2006

An early step in the transposition of bacteriophage Mu DNA in vitro is a DNA strand-transfer reaction that generates an intermediate DNA structure in which the Mu donor DNA and the target DNA are covalently joined. DNA replication, initiated at the DNA forks in this intermediate, generates a cointegrate product; simple insert products can also be formed from the same intermediate by degradation of a specific segment of the structure, followed by gap repair. This DNA strand-transfer reaction requires ATP, magnesium, the Mu A and Mu B proteins, and a factor supplied by an Escherichia coli cell extract. We have now shown that the host protein factor requirement can be satisfied by purified protein HU. The defined system has been used to determine the DNA substrate requirements for the reaction. The reaction requires the two Mu ends, located on the same DNA molecule, in the same relative orientation to one another as in the phage Mu genome. To participate in the strand-transfer reaction efficiently the mini-Mu plasmid, used as the transposon donor, must be supercoiled; the target DNA molecule may be supercoiled, relaxed circular, or linear.
Polymorphism and evolution of Alu sequences in the human low density lipoprotein receptor gene.
Authors: Hobbs H H , Lehrman M A , Yamamoto T , Russell D W
Published: May 2006

Two clusters of Alu sequences in the human low density lipoprotein (LDL) receptor gene have been analyzed in detail. One Alu cluster is present within the intron separating exons 15 and 16 of the gene and contains a polymorphic Pvu II site. The presence or absence of this site gives rise to two allelic fragments of 14 and 16.5 kilobases, respectively, in genomic Southern blots using cloned cDNA probes. This DNA polymorphic site is caused by a single adenine to guanine transition within an Alu repetitive element. The second cluster of Alu sequences is located in exon 18 of the LDL receptor gene. Southern blotting of primate DNAs suggests that this cluster became associated with the gene about 30 million years ago. Comparison of bovine DNA sequences, which lack this Alu cluster, with those of the human indicates that the Alu sequences inserted in exon 18 in two independent events.
Alleles of the Ly-17 alloantigen define polymorphisms of the murine IgG Fc receptor.
Authors: Holmes K L , Palfree R G , Hammerling U , Morse H C
Published: May 2006

Antibodies specific for allelic determinants of the cell membrane alloantigen Ly-17 were found to react with genetically determined polymorphic sites on the murine IgG Fc receptor (Fc gamma R). Monoclonal antibodies specific for Ly-17.2 and the Fc gamma R detected identical populations of cells in thymus, spleen, lymph node, and bone marrow and had nearly identical reactivity with a large number of hematopoietic neoplasms. Antibodies to the Fc gamma R and either allele of Ly-17 blocked binding of rabbit IgG dimers to the Fc gamma R on spleen and bone marrow cells. Antibodies to the Fc gamma R also blocked binding of antibodies to either allele of Ly-17, whereas anti-Ly-17 antibodies only partially blocked the binding of antibodies to the Fc gamma R. Biochemical studies showed that antibodies to Ly-17 and the Fc gamma R precipitated proteins of Mr 55,000-60,000. The identity of the proteins recognized by these antibodies was confirmed by sequential immunoprecipitation.
Stabilization of the long central helix of troponin C by intrahelical salt bridges between charged amino acid side chains.
Authors: Sundaralingam M , Drendel W , Greaser M
Published: May 2006

The unusual dumbbell shape of troponin C is due to the presence of a long alpha-helix of nine turns that connects the amino- and carboxyl-terminal calcium-binding domains. The center of the long helix appears to be stabilized by several salt bridges. The long helix is also bent about 16 degrees at glycine-92. Calmodulin, which lacks the central glycine, also is predicted to be stabilized by salt bridges in the central helix. The presence of a proline residue in the center of the long helix of ascidian troponin C and the myosin regulatory light chains suggests that a sharper bend may occur in these molecules. The conservation of the bend and salt bridges in the related calcium-binding proteins suggests they may have an important biological function. The structure of troponin C suggests that intrahelix salt bridges between neighboring charged residues may be involved in the stabilization of protein secondary structure. The preponderance of potential salt bridges in other muscle proteins as well may be related to their elongated structures and their participation in the contractile process.
Cytotactin, an extracellular matrix protein of neural and non-neural tissues that mediates glia-neuron interaction.
Authors: Grumet M , Hoffman S , Crossin K L , Edelman G M
Published: May 2006

An extracellular matrix protein, cytotactin, with widespread tissue distribution has been identified, isolated, and partially characterized. Cytotactin mediates glia-neuron adhesion in vitro, but unlike Ng-CAM, the neuron-glia cell-adhesion molecule, it is absent from neurons. Cytotactin was isolated from 14-day embryonic chicken brains as structurally related polypeptides of Mr 220,000, 200,000, and 190,000. These polypeptides were efficiently extracted in the absence of detergent and appeared to be disulfide-linked into higher polymers. Immunofluorescence staining with specific antibodies indicated that cytotactin is found in extracellular spaces and in basement membranes of a variety of non-neural tissues including smooth muscle, lung, and kidney. In the cerebellum, it appears on glial end-feet, on Bergmann glial fibers, and in extracellular spaces. The molecule is synthesized by glia and cells from smooth muscle, lung, and kidney. It is found at the surface of glia in culture in a cell-associated fibrillar pattern. A survey of the times and sites of its appearance during embryogenesis is consistent with the hypothesis that cytotactin is a cell-substrate adhesion molecule that may mediate cell migration in a site-restricted fashion.
Polymorphic restriction endonuclease sites linked to the HLA-DR alpha gene: localization and use as genetic markers of insulin-dependent diabetes.
Authors: Stetler D , Grumet F C , Erlich H A
Published: May 2006

Polymorphic restriction endonuclease sites within the HLA-DR alpha gene have been defined, localized, and used as genetic markers in the analysis of susceptibility to insulin-dependent diabetes mellitus (IDDM). Hybridization of Bgl II-digested human genomic DNA with a cDNA clone for the HLA-DR alpha chain (pDR alpha-1) has revealed three allelic restriction fragment lengths: 3.8 kilobase pairs (kb), 4.2 kb, and 4.5 kb. Hybridization of EcoRV-digested human genomic DNA with the same probe has revealed two allelic polymorphic restriction fragment lengths: 9.2 kb and 13.0 kb. By analysis of double digests of genomic DNA from individuals homozygous for each of the allelic variants, the polymorphic restriction sites were found to be clustered near the 3' end of the HLA-DR alpha gene. The observed correlations of DR alpha Bgl II restriction site variants with serologically determined DR specificities suggest linkage disequilibrium between the DR alpha and DR beta loci. The 3.8-kb fragment is correlated with the DR1 type (Pc = 4.4 X 10(-4)); and the 4.2-kb fragment, with a subset (B8,DR3) of the DR3 type (Pc = 5.1 X 10(-4)) and with the DR6 type. The segregation pattern of HLA-DR alpha polymorphic Bgl II restriction fragments was analyzed in six IDDM families. The observed association of IDDM with the Bgl II 4.2-kb DR alpha restriction variant is higher than with existing serological markers and supports the utility of this approach in elucidating IDDM inheritance.
Distinct H-2-linked regulation of T-cell responses to the pre-S and S regions of the same hepatitis B surface antigen polypeptide allows circumvention of nonresponsiveness to the S region.
Authors: Milich D R , McNamara M K , McLachlan A , Thornton G B , Chisari F V
Published: May 2006

Recently, additional polypeptide components of the surface envelope of hepatitis B virus (HBV) have been identified. The pre-S(1) and pre-S(2) regions of the HBV genome encode NH2-terminal amino acid residues that together with the S-gene product (25 kDa) comprise polypeptides of 33 kDa and 39 kDa. The possible immunopathologic significance of these larger polypeptides and their relevance to vaccine development prompted us to examine the murine immune response to pre-S(2)-encoded determinants as compared to S-encoded determinants on the same polypeptide. Previous work showed that the pre-S(2) region elicits greater antibody production in vivo than does the S region of hepatitis B surface antigen. In this study, we examined immunogenicity of the pre-S(2) region at the T-cell level, H-2- and non-H-2-linked genetic influences on the pre-S(2) response, and the effect of the immune response to one region on the immune response to the other region. The results indicate that (i) the pre-S(2) region is significantly more immunogenic than the S region at the T-cell level; (ii) pre-S(2)-region-specific T-cell activation is regulated by H-2-linked genes and correlates with the H-2 restriction of in vivo antibody production to the pre-S(2) region; (iii) the H-2 restriction of the T-cell response to the pre-S(2) region is distinct from the H-2 restriction of the T-cell response to S-region determinants; (iv) non-H-2-linked and non-Igh-linked genes also influence the humoral immune response to the pre-S(2) region; and (v) immunization of an S-region-nonresponder, pre-S(2)-region T-cell-responder strain with HBV envelope particles containing both the pre-S(2) and S regions can circumvent nonresponsiveness to the S region through pre-S(2)-specific T-cell helper function.
Intravenous cyclosporine activates afferent and efferent renal nerves and causes sodium retention in innervated kidneys in rats.
Authors: Moss N G , Powell S L , Falk R J
Published: May 2006

The effect of acute intravenous infusion of cyclosporine (10 mg/kg) on efferent renal and genitofemoral nerve activity and afferent renal nerve activity was studied in anesthetized rats. All animals were studied after unilateral renal denervation and extracellular fluid volume expansion. Activity of both efferent sympathetic nerves was increased significantly by cyclosporine infusion (renal, 69%; genitofemoral, 60%). Afferent renal nerve activity was increased 82% after cyclosporine (P less than 0.05). Urine flow rate and both absolute and fractional sodium excretion from the innervated kidney were reduced 50% after cyclosporine infusion (P less than 0.01). Absolute and fractional sodium excretion from the denervated kidney were significantly increased after cyclosporine. Infusion of vehicle had no significant effect on any measured variable in innervated or denervated kidneys. These studies demonstrate the capacity of cyclosporine to increase efferent sympathetic nerve activity and afferent nerve activity. It is also shown that sodium retention resulting from acute infusion of cyclosporine can be attributed to the increase in efferent renal nerve activity.
Evidence for functional interaction between domains II and V of 23S ribosomal RNA from an erythromycin-resistant mutant.
Authors: Douthwaite S , Prince J B , Noller H F
Published: May 2006

A mutation affording low levels of erythromycin resistance has been obtained by in vitro hydroxylamine mutagenesis of a cloned ribosomal RNA operon from Escherichia coli. The site of the mutational event responsible for antibiotic resistance was localized to the gene region encoding domain II of 23S rRNA by replacement of restriction fragments in the wild-type plasmid by corresponding fragments from the mutant plasmid. DNA sequencing showed that positions 1219-1230 of the 23S rRNA gene are deleted in the mutant. Since all previously characterized rRNA mutations conferring resistance to erythromycin show changes exclusively in domain V, our present findings provide direct evidence for functional interaction between domains II and V of 23S rRNA.
Gene gating: a hypothesis.
Authors: Blobel G
Published: May 2006

It is assumed that the genome of a higher eukaryotic organism is organized into a number of distinct three-dimensional (3-D) structures, each characteristic for a given differentiated state. These discrete 3-D structures are envisioned to develop in a hierarchical and largely irreversible manner from an omnipotent 3-D structure of the zygotic genome. The information for these processes is assumed to reside in the genome. The nuclear pore complexes, the peripheral nuclear lamina, and components of the nuclear core are proposed to be among the topologically most proximal organelles that interpret this information and thereby serve in the maintenance and the alteration of the 3-D structure of the genome during development, differentiation, and the cell cycle. The nuclear pore complexes are envisioned to serve as gene-gating organelles capable on interacting specifically with expanded (transcribable) portions of the genome. Their nonrandom distribution on the nuclear surface would reflect the underlying periodic organization of the genome into expanded and compacted domains, alternating with each other. All transcripts of a given gated gene would leave the nucleus by way of that pore complex to which the gene is gated. Implications for cell asymmetry and polarity are discussed and evolutionary considerations are presented.
Hepatic lipocytes: the principal collagen-producing cells of normal rat liver.
Authors: Friedman S L , Roll F J , Boyles J , Bissell D M
Published: May 2006

Hepatic lipocytes were isolated from normal rat liver and established in culture. A virtually pure isolate was obtained by fractionating enzymatically digested liver on a discontinuous gradient of arabinogalactan. Isolated cells displayed prominent rough endoplasmic reticulum and typical cytoplasmic droplets containing vitamin A. Lipocytes in primary culture were shown by immunofluorescence to secrete collagen types I, III, and IV and also laminin. Immunoassay of culture media showed that lipocytes during the first week in culture secrete type I (72.1-86.2% of total measured soluble collagen), type III (2.6-7.2%), and type IV (11.2-29.7%) collagens. Five percent of total secreted protein was collagen compared with 0.2% in similarly cultured hepatocytes and 1.7% in sinusoidal endothelial cells, as measured by the production of peptide-bound [3H]hydroxyproline in cells incubated with [3H]proline. The calculated amount of collagen synthesized by lipocytes per microgram of cellular DNA was 10-fold greater than that produced by hepatocytes and over 20-fold greater than that produced by endothelial cells. The findings indicate that collagen synthesis and secretion are specialized functions of hepatic lipocytes, and that, in cells from normal liver, this represents production primarily of type I collagen. The phenotypic resemblance of these cells to fibroblasts supports the hypothesis that lipocytes are a major source of collagen in pathologic fibrosis and may be precursors of the fibroblast-like cells observed in liver injury.
Determination of the primary amino acid sequence specifying the alpha-bungarotoxin binding site on the alpha subunit of the acetylcholine receptor from Torpedo californica.
Authors: Wilson P T , Lentz T L , Hawrot E
Published: May 2006

A region of the alpha subunit of the nicotinic acetylcholine receptor containing the alpha-bungarotoxin-binding domain was mapped on the primary amino acid sequence in relation to asparagine-141, the presumed site of N-linked glycosylation. Proteolytic fragments of the alpha subunit, immobilized onto positively charged membrane filters, that bind 125I-labeled bungarotoxin were further analyzed on the basis of the size of the fragments and the presence of asparagine-141 as determined by susceptibility to digestion with endoglycosidase H. The bungarotoxin-binding site was found not to reside between amino acid residues 1 and 140 since bungarotoxin-binding fragments that are considerably larger than 140 amino acids and lack N-linked oligosaccharide chains were detected. The size of the smallest bungarotoxin-binding fragment containing asparagine-141 and the size of fragments produced by digestion with V8 protease further indicated that the bungarotoxin-binding site is contained within amino acid residues 153-241. A 32-amino acid synthetic peptide comprising a portion of this region (residues 173-204) was tested for its ability to bind 125I-labeled bungarotoxin. 125I-labeled bungarotoxin bound to the peptide and was competed by unlabeled bungarotoxin and d-tubocurarine with IC50 values of 0.5 microM and 2 mM, respectively. We conclude that a major determinant of the bungarotoxin-binding site on the alpha subunit resides between residues 173 and 204.
Vasoactive intestinal polypeptide acts synergistically with norepinephrine to depress spontaneous discharge rate in cerebral cortical neurons.
Authors: Ferron A , Siggins G R , Bloom F E
Published: May 2006

Cortical neurons are densely innervated by noradrenergic fibers and by intrinsic cortical interneurons containing vasoactive intestinal polypeptide (VIP). Biochemically, VIP and norepinephrine (NE) synergistically interact to stimulate the synthesis of cyclic AMP in cortical slices. Therefore, we sought physiological indices of this peptide-monoamine interaction by applying VIP and NE to single cortical neurons of the rat while recording their spontaneous discharge. VIP applied alone inhibited discharge of 24% and accelerated discharge in 20% of cortical neurons. NE alone had a predominantly depressant effect on the same neurons. However, when VIP was retested during the continuous application of subthreshold currents of NE, VIP exerted predominantly depressant effects. These synergistic inhibitions resulted even in cells previously showing excitations to VIP alone. If VIP alone was depressant, subthreshold NE further enhanced the VIP depression. Subthreshold amounts of phenylephrine, an alpha-adrenoceptor agonist, also produced comparable interactions, suggesting involvement of an alpha receptor, as in the biochemical studies. These results support a peptide-monoamine interaction in cortex that could have important ramifications for neuronal integration.
Cloned cDNA for rabbit erythrocyte carbonic anhydrase I: A novel erythrocyte-specific probe to study development in erythroid tissues.
Authors: Konialis C P , Barlow J H , Butterworth P H
Published: May 2006

Present understanding of gene expression in erythropoietic tissues is derived solely from studies of the globin genes. Of the three distinct carbonic anhydrase (carbonate dehydratase; carbonate hydro-lyase, EC 4.2.1.1) isozymes, carbonic anhydrase I is erythrocyte-specific and, in humans, is under developmental control. The appearance of carbonic anhydrase I in the erythrocyte late in fetal life follows closely the gamma- to beta-globin switch. In order to study the expression of this erythrocyte-specific nonglobin protein, we set out to isolate a cloned carbonic anhydrase I cDNA. A mixture of 17-base-long synthetic oligonucleotides was used as an in situ hybridization probe to screen a rabbit reticulocyte cDNA library. Two clones were isolated, and the complete nucleotide sequence of the clone with the largest insert was determined and shown to code for carbonic anhydrase I. This clone, designated pRCAI, is near full length and has provided the 40% of the amino acid sequence of rabbit carbonic anhydrase I, which was not known hitherto. The deduced primary structure has revealed potentially significant changes in the vicinity of the active site of the rabbit carbonic anhydrase I when compared with carbonic anhydrase I and II sequences from other species.
Active site structures of deoxyhemerythrin and oxyhemerythrin.
Authors: Stenkamp R E , Sieker L C , Jensen L H , McCallum J D , Sanders-Loehr J
Published: May 2006

The physiologically active forms of the nonheme-iron, oxygen-transport protein hemerythrin have been studied by x-ray crystallographic techniques. At 3.9-A resolution, a difference electron-density map between the deoxy form and met form (methemerythrin) of the protein suggests only small differences in the binuclear iron complexes. The coordination of the iron atoms appears to be the same in both the deoxy and met forms, one iron of the complexes being pentacoordinate, the other iron being hexacoordinate. The iron atoms appear to be somewhat farther apart in the deoxy form. A 2.2-A resolution study of oxyhemerythrin shows that dioxygen binds to one iron atom--the pentacoordinate one in the met form of the protein, the same binding site found for azide in azidomethemerythrin.
Involvement of ribosomal protein L7/L12 in control of translational accuracy.
Authors: Kirsebom L A , Isaksson L A
Published: May 2006

The effects of two mutations, which map at the rplL locus and both give a changed 50S ribosomal protein L7/L12, were studied. Both mutations are associated with an increased misreading of all three nonsense codons in vivo and ribosomes from the mutants give an increased misreading of the phenylalanine codon UUU by tRNALeu in vitro. The rplL-associated misreading in vitro is not limited to a particular type of mRNA or tRNA. Results from a translational proofreading assay, using mutant ribosomes, suggest that protein L7/L12 is involved in the control of translational accuracy by contributing to the efficiency of a translational proofreading step(s).
Molecular cloning and complete sequence determination of RNA genome of human rhinovirus type 14.
Authors: Callahan P L , Mizutani S , Colonno R J
Published: May 2006

The genomic RNA of human rhinovirus type 14 was cloned in Escherichia coli and the complete nucleotide sequence was determined. The RNA genome is 7212 nucleotides long. A single large open reading frame of 6536 nucleotides was identified, which starts at nucleotide 678 and ends 47 nucleotides from the 3' end of the RNA genome. Comparisons of the specified proteins with those of other picornaviruses showed a striking homology (44-65%) between rhinovirus and poliovirus. The rhinovirus genomic RNA is rich in adenosine (32.1%) and strongly favors an adenosine or uridine in the third position of codons. The predicted map locations of all the rhinovirus structural and non-structural proteins and their proposed proteolytic cleavage sites are described.
A cosmid for selecting genes by complementation in
Aspergillus nidulans
: Selection of the developmentally regulated
yA
locus
Authors: Yelton M. Melanie , Timberlake William E. , Hondel C. A. M. J. J. van den
Published: May 2006

We constructed a 9.9-kilobase cloning vector, designated pKBY2, for isolating genes by complementation of mutations in
Aspergillus nidulans
. pKBY2 contains the bacteriophage λ
cos
site, to permit
in vitro
assembly of phage particles; a bacterial origin of replication and genes for resistance to ampicillin and chloramphenicol, to permit propagation in
Escherichia coli
; the
A. nidulans trpC
+
gene, to permit selection in
Aspergillus
; and a unique
Bam
HI restriction site, to permit insertion of DNA fragments produced by digestion with restriction endonucleases
Bam
HI,
Bgl
II,
Mbo
I, or
Sau
3A. We used this cosmid to form a quasirandom recombinant DNA library containing 35- to 40-kilobase DNA fragments from a wild-type strain of
A. nidulans
. DNA from this library transformed a yellow-spored (
yA
-
)
pabaA
-
trpC
-
Aspergillus
strain (FGSC237) to
trpC
+
at frequencies of approximately 10 transformants per μg of DNA. Three of approximately 1000
trpC
+
pabaA
-
colonies obtained were putative
yA
+
transformants, because they produced wild-type (green) spores. DNA from each of the green-spored transformants contained pKBY2 sequences and DNA from two transformants transduced
E. coli
to ampicillin resistance following treatment
in vitro
with a λ packaging extract. The cosmids recovered in
E. coli
had similar restriction patterns and both yielded
trpC
+
transformants of
A. nidulans
FGSC237, 85% of which produced green spores. Several lines of evidence indicate that the recovered cosmids contain a wild-type copy of the
yA
gene.
Antibodies to peptides corresponding to a conserved sequence of gonococcal pilins block bacterial adhesion.
Authors: Rothbard J B , Fernandez R , Wang L , Teng N N , Schoolnik G K
Published: May 2006

Antisera generated against each of seven synthetic peptides corresponding to constant and variable sequences of the pilin from gonococcal strain MS11 were assayed for their ability to crossreact with intact pili from both homologous and heterologous strains. The peptides elicited roughly equal antipeptide responses but varied substantially in their ability to elicit antisera that crossreacted with intact pili. Of the antisera to peptides corresponding to regions of conserved sequence, antisera directed against residues 69-84 were the most efficient in binding pili from all strains tested in both solid-phase assays and immunoblots. Anti-69-84 also efficiently precipitated a tryptic fragment of pilin known to bind human endocervical cells. Sera against the two peptides (121-134 and 135-151) previously shown to contain strain-specific epitopes crossreacted with MS11 pili equally well, but differed in their ability to bind pili from heterologous strains. Anti-121-134 was strain-specific whereas anti-135-151 bound all pilin tested. Each of the sera was examined for its ability to inhibit bacterial adhesion to a human endometrial carcinoma cell line. Sera generated against residues 41-50 and 69-84 successfully inhibited a heterologous gonococcal strain from binding. These peptides could be important components of an effective vaccine for the prevention of gonorrhea.
Interaction of the lambda site-specific recombination protein Xis with attachment site DNA.
Authors: Yin S , Bushman W , Landy A
Published: May 2006

Nuclease protection experiments show that Xis protein of bacteriophage lambda specifically binds attachment (att) site DNA. The region of Xis binding, present in both the phage att site and the right prophage att site, extends from position -102 to position -62 in the P arm. The sequence of this region, the positions of purines protected by Xis against methylation, and the binding of Xis to a resected att site indicate the presence of two binding sites. The postulated recognition elements, contained in 13-base-pair direct repeats separated by 7 base pairs, are situated on the same face of the DNA helix. Protection experiments performed with DNase I suggest that the DNA wraps around (or along the surface of) the bound Xis protein. The Xis binding data presented here establishes that Xis, like the other two proteins involved in lambda site-specific recombination, interacts specifically with att DNA. This rules out that class of models in which the profound effects of Xis on the directionality of site-specific recombination are mediated solely through protein-protein interactions or modification of another protein. In addition, nuclease protection experiments with pairwise combinations of the proteins show that Xis and integration host factor (IHF), or Xis and Int, can bind simultaneously to either the phage or right prophage att sites, and the DNA sequences protected are the sum of those protected with each protein alone. It is therefore unlikely that the effect of Xis on the direction of recombination is exerted by directly blocking the binding of Int or IHF to one or more of their respective binding sites.
Altered Cro repressors from engineered mutagenesis of a synthetic cro gene.
Authors: Eisenbeis S J , Nasoff M S , Noble S A , Bracco L P , Dodds D R , Caruthers M H
Published: May 2006

A portion of the gene coding for the Cro repressor protein of bacteriophage lambda has been chemically synthesized, incorporating base pair changes that generate restriction endonuclease sites without altering the amino acid coding sequence. These restriction endonuclease sites were used to remove small segments of the synthetic cro gene and the segments were replaced with duplexes carrying desired mutations. Altered Cro proteins produced by mutants constructed in this manner were then assayed for binding to lambda operator OR3 in vivo. Mutations directed into the region of the cro gene encoding the alpha-3 helix produced altered Cro proteins with a range of affinities for operator DNA. These changes suggest which amino acids play an important role in Cro-OR3 complex formation.
Monte Carlo study of the GTP cap in a five-start helix model of a microtubule.
Authors: Chen Y D , Hill T L
Published: May 2006

Earlier Monte Carlo studies on a single-helix model of the GTP cap at the end of a microtubule are extended here to a more realistic five-start helix model of the microtubule end. As in the earlier work, phase changes occur at the microtubule end: the end is either capped with GTP and growing slowly or uncapped and shortening rapidly, and these two regimes alternate (at a given tubulin concentration) at steady state. Macroscopic rate constants for the two-phase model are deduced from the Monte Carlo results. The macroscopic rate constants lead to properties that are in semiquantitative agreement with related experiments of Mitchison and Kirschner.
Immunologic abnormalities of mice bearing the gld mutation suggest a common pathway for murine nonmalignant lymphoproliferative disorders with autoimmunity.
Authors: Davidson W F , Holmes K L , Roths J B , Morse H C
Published: May 2006

Mice bearing the autosomal recessive mutation gld have been shown to develop massive lymphadenopathy, hypergammaglobulinemia, and autoantibodies and to die prematurely with interstitial pneumonitis. In this study, lymphocytes from C3H gld and C3H +/+ mice were examined for a variety of phenotypic and functional characteristics. Spleens and lymph nodes of mutant mice were expanded by an aberrant population of Ly-5(B220)+ surface immunoglobulin negative cells that were Thy-1+Ly-1+ or Thy-1-Ly-1+. Cells from both tissues of mutant mice older than 8 wk were impaired in their ability to proliferate in response to allogeneic stimuli, and supernatants of cells stimulated with concanavalin A contained significantly reduced levels of interleukin 2. Cytotoxic T-lymphocyte responses of spleen and lymph node cells from C3H gld mice were normal at all ages tested. These results are strikingly similar to those obtained with C3H mice homozygous for the nonallelic autosomal recessive mutation lpr. We suggest that the similarities between the syndromes induced by these two mutations may reflect alterations in different enzymes that act in a common metabolic pathway of major importance to the differentiation and function of T cells.
Transfer RNA is an essential component of the ubiquitin- and ATP-dependent proteolytic system.
Authors: Ciechanover A , Wolin S L , Steitz J A , Lodish H F
Published: May 2006

Protein degradation via the nonlysosomal ATP-dependent pathway in rabbit reticulocytes involves a number of components. In the initial event, ubiquitin, an abundant 76-residue polypeptide, becomes covalently linked to the protein substrate in an ATP-requiring reaction. Once marked in this way, the conjugated protein is proteolyzed in a reaction that also requires ATP. Ubiquitin-marking appears to be important to the progression of cells from one stage to another of the cell cycle; it may also be involved in gene activation. Here we show that tRNA is another essential component of the system. Ribonucleases strongly inhibit the ubiquitin- and ATP-dependent degradation of 125I-labeled bovine serum albumin in the reticulocyte system in vitro. RNAs extracted from fractions of the reticulocyte extract or from mouse cells restore proteolytic activity. When the RNA is fractionated by gel electrophoresis, only the tRNA fraction is active in restoring proteolysis. Furthermore, pure mouse tRNAHis, isolated by immunoprecipitation with patient autoimmune sera, restores the proteolytic activity. The possibility that the level of uncharged tRNA in mammalian cells regulates the ubiquitin- and ATP-dependent proteolytic system is discussed.
Regulated expression of endonuclease EcoRI in Saccharomyces cerevisiae: nuclear entry and biological consequences.
Authors: Barnes G , Rine J
Published: May 2006

In an investigation to determine how proteins are localized within the nucleus of a cell, we demonstrate that the restriction endonuclease EcoRI is able to enter and function within the nucleus of Saccharomyces cerevisiae when this prokaryotic protein is synthesized in vivo. The EcoRI endonuclease was produced in yeast under the transcriptional control of a regulated yeast promoter by ligating a DNA fragment containing only coding sequences for the endonuclease to the promoter element of the yeast GAL1 gene (the structural gene for galactokinase, EC 2.7.1.6). Yeast cells harboring a plasmid containing this promoter-gene fusion are able to grow under conditions that repress transcription from the GAL1 promoter. However, under inducing conditions, these yeast cells are unable to grow. Moreover, rad52 mutants, which are deficient in the repair of double-strand breaks, are more sensitive to the presence of the promoter-gene fusion plasmid than are wild-type cells. We demonstrate that the EcoRI endonuclease activity is present in lysates prepared from yeast transformants grown under conditions that induce transcription of GAL1, but this activity is not detectable in cells grown under conditions that repress transcription from the promoter. Furthermore, analysis of yeast chromosomal DNA shows that the endonuclease enters the yeast nucleus and cleaves DNA specifically at EcoRI recognition sites.
Independent control elements that determine yolk protein gene expression in alternative Drosophila tissues.
Authors: Garabedian M J , Hung M C , Wensink P C
Published: May 2006

The adjacent and divergently transcribed yp1 and yp2 genes of Drosophila were separated at a site that is 342 base pairs upstream of yp2 and 883 base pairs upstream of yp1. Each gene was separately used to transform Drosophila germ-line-producing flies with a single copy of the introduced gene. Transcripts from the introduced genes were found only in adult females. Thus, the introduced genes maintain the sex- and time-specific expression pattern of the endogenous yp genes. However, the pattern of tissue-specific expression differed among the two introduced genes and the endogenous genes. Transcripts from both of the endogenous genes are found in fat bodies and ovaries. In contrast, transcripts from the introduced yp1 gene are found only in fat bodies, and those from the introduced yp2 gene are found only in ovaries. Thus, each introduced DNA segment lacks at least one of the cis-acting elements required for the normal pattern of tissue-specific expression. These results indicate that the expression of a yp gene in different tissues is determined by different cis-acting elements.
Efficient induction and selection of chloroplast-encoded antibiotic-resistant mutants in
Nicotiana
Authors: Fluhr Robert , Aviv Dvora , Galun Esra , Edelman Marvin
Published: May 2006

A high rate of plastome-encoded mutations was induced in
Nicotiana
by exposing seeds to
N
-nitroso-
N
-methylurea. Seeds then were subjected to nutritional and
in vitro
selection procedures for systematic isolation of plastome-dependent antibiotic-resistant plants. Multiple flowering lines resistant to streptomycin, spectinomycin, lincomycin, or chloramphenicol were obtained. A detailed analysis of the streptomycin-resistant lines is presented. Sexual hybridization, cybrid formation following protoplast fusion, and
in organello
protein synthesis were used to rigorously assign the mutations to the chloroplast genome. The efficient rates of mutagenesis combined with the
in vitro
mass-screening procedures described here should facilitate investigation of fundamental aspects of chloroplast genetics in higher plants.
Strong antitumor activities of IgG3 antibodies to a human melanoma-associated ganglioside.
Authors: Hellström I , Brankovan V , Hellström K E
Published: May 2006

Three mouse monoclonal IgG3 antibodies, 2B2, IF4, and MG-21, recognize a GD3 ganglioside antigen that is expressed at the cell surface of most human melanomas. All three antibodies mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro when tested with human lymphocytes as effector cells in a 2-hr or 4-hr 51Cr-release test, and one antibody, MG-21, also gives strong complement-dependent cytotoxicity with human serum. Antibody 2B2, which gives ADCC also in the presence of mouse lymphocytes, inhibited the outgrowth of a human melanoma in nude mice, but antibody IF4, which showed no ADCC with mouse lymphocyte effectors, did not.
Characterization of a protein, released by the T47D cell line, immunologically related to the major envelope protein of mouse mammary tumor virus.
Authors: Segev N , Hizi A , Kirenberg F , Keydar I
Published: May 2006

The T47D human mammary adenocarcinoma cell line in vitro releases viral particles as well as soluble proteins. Both fractions were shown to contain antigens that immunologically crossreact with the major glycoprotein (gp52) of mouse mammary tumor virus. The crossreacting antigens are located on polypeptides with apparent molecular weights of about 68,000 and 60,000. The larger one is present in viral particles whereas both are found in the soluble fraction. Both proteins are glycosylated. The human tissue culture proteins differ from gp52 not only in molecular weight but also in charge heterogenity and in polypeptide profiles obtained after partial proteolysis. The results suggest that there is a restricted similarity between MMTV gp52 and the immunologically related T47D proteins.
Expression of the metastatic phenotype in cells transfected with human metastatic tumor DNA.
Authors: Bernstein S C , Weinberg R A
Published: May 2006

NIH 3T3 mouse fibroblasts form nonmetastasizing fibrosarcomas upon transformation by the Ha-ras oncogene isolated from the EJ human bladder carcinoma cell line and subcutaneous inoculation into immunocompetent NFS/NCr mice. DNA from a human metastatic tumor was transfected into these Ha-ras transformants, and one of the resulting colonies yielded a lung metastasis after subcutaneous inoculation. DNA was isolated from this metastasis and subjected to a second round of transfer into Ha-ras-transformed NIH 3T3 cells. Inoculation of these transfected cultures into mice led once again to formation of metastases, this time at a higher frequency. Examination of four of the resulting metastases revealed discrete human DNA fragments that were common to all four. These findings demonstrate that the metastatic phenotype can be transferred via DNA from cell to cell and is associated with the presence of a discrete DNA segment. This segment is not identical to the myc oncogene or to any of the frequently detected ras tumor oncogenes.
Transposition of Tc1 in the nematode Caenorhabditis elegans.
Authors: Eide D , Anderson P
Published: May 2006

We have identified a strain of Caenorhabditis elegans in which the transposable element Tc1 is genetically active. Most spontaneous mutations affecting the unc-54 myosin heavy chain gene of C. elegans variety Bergerac are due to insertions of Tc1 within unc-54. The Bergerac genome contains an unusually high number of Tc1 elements, but this is not responsible for transpositional activity. Another variety of C. elegans, strain DH424, contains an equally high number of Tc1 elements, but transpositions are not detected. Tc1 insertion mutations are genetically unstable. They revert to unc-54+ in both germ-line and somatic cells. Germ-line revertants are wild type and contain precise or nearly precise excisions of Tc1. Somatic revertants are genetic mosaics; they contain small patches of revertant muscle tissue in otherwise mutant animals. The pattern of mosaicism often allows us to know when and where during muscle development the excisions occur. Somatic reversion can be over 1000-fold more frequent than germ-line reversion.
Relationship of M protein genes in group A streptococci.
Authors: Scott J R , Pulliam W M , Hollingshead S K , Fischetti V A
Published: May 2006

The M protein of group A streptococci is considered one of the primary virulence determinants of this organism because it prevents phagocytosis. All of the 56 different M protein types of group A streptococci tested contain a DNA region that hybridizes with a labeled probe consisting of DNA encoding the type 6 M protein (emm6 gene). In addition, 2 of 3 functionally M- group A strains (strains that are successfully phagocytized) also contain this gene, but this DNA is not present in 1 M- strain. Of the other streptococcal groups, only groups C and G, which are closely related to group A, contain DNA that hybridizes with emm6. None of the non-streptococcal Gram-positive bacteria tested contains emm6-related DNA. In the strain from which emm6 was cloned, there appears to be only a single copy of this gene.