bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure and nucleotide sequence of the heavy chain gene of HLA-DR.
Authors: Das H K , Lawrance S K , Weissman S M
Published: May 2006

We have used a 175-nucleotide-long primer extension product corresponding to the 5' end of HLA-DR alpha-chain mRNA to isolate a genomic clone from a human DNA library. The entire HLA-DR alpha gene is contained in two contiguous EcoRI fragments spanning about 7.5 kilobases (kb); most of the sequence has been determined. The 5' end of the gene is contained in a 4.4-kb fragment, and the coding segments and the 3' untranslated region are contained in a 3.1-kb fragment. The gene is split into five exons. The 5' untranslated region, the leader peptide, and the first two NH2-terminal amino acids are fused into the first exon. Exons 2 and 3 represent two extracellular coding domains of mature p34. The transmembrane domain, cytoplasmic domain, and part of the 3' untranslated region are merged into a fourth exon. The rest of the 3' untranslated region is in exon 5. The predicted amino acid sequence of mature p34, as deduced from its gene structure, has 229 residues and reveals a single potential disulfide loop (between cysteine residues 107 and 163) as well as a 22-amino acid residue membrane integrated segment (residues 193-214). Fifteen amino acids (residues 215-229) reside on the cytoplasmic side of the plasma membrane. There is considerable amino acid sequence homology between the second external domains of p34 and p29, as well as the immunoglobulin-like third domain of HLA-B7, and beta 2-microglobulin and the homologous constant region domains of the light and heavy chains of immunoglobulins.
Depolarization and increased conductance precede superoxide release by concanavalin A-stimulated rat alveolar macrophages.
Authors: Cameron A R , Nelson J , Forman H J
Published: May 2006

Rat alveolar macrophages release superoxide into the extracellular medium when stimulated by concanavalin A. This process, the respiratory burst, is characterized by a delay between binding of the stimulus and release of superoxide. It has been proposed that a key event that occurs during this delay period is the alteration of membrane electrical potential. Microelectrode impalement was used to directly measure electrical properties of the plasma membrane. Upon addition of concanavalin A, the membrane potential depolarized 21%, and membrane electrical resistance decreased 16%. Parallel chemical measurement of superoxide release indicated that these changes in electrical properties precede the release of superoxide.
Human cytomegalovirus (strain AD169) contains sequences related to the avian retrovirus oncogene v-myc.
Authors: Spector D H , Vacquier J P
Published: May 2006

We have detected nucleotide sequences related to the transforming gene (v-myc) of avian myelocytomatosis virus MC-29 in the DNA genome of human cytomegalovirus (HCMV) strain AD169. Cloned DNA representing the entire 1.5-kilobase-pair oncogene v-myc and subfragments of this gene were hybridized to EcoRI-cleaved HCMV virion DNA and cloned subgenomic HCMV DNA fragments. Only a 0.5-kilobase-pair Pst I-Sal I subfragment representing the 5' end of the coding sequence of the v-myc oncogene hybridized to the HCMV DNA. We have localized these v-myc-related sequences to five regions in the long unique segment of the HCMV genome corresponding to EcoRI fragments C, I, P, R, and b and to regions within the EcoRI junction fragments F and H at or near the repeats bounding the short unique segment. There was no hybridization of these HCMV sequences to other retroviral oncogenes tested including v-src, v-myb, v-erb, v-ST-fes, v-fos, v-ras (Harvey), v-mos, and v-abl.
Biphasic regulation by dibutyryl cyclic AMP of tubulin and actin mRNA levels in neuroblastoma cells.
Authors: Ginzburg I , Rybak S , Kimhi Y , Littauer U Z
Published: May 2006

Blot hybridization analysis that used labeled tubulin cDNA probes revealed that N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate [dibutyryl cyclic AMP (Bt2cAMP)] initially increases and later decreases the level of tubulin mRNA in a neuroblastoma-glioma hybrid cell line as well as in the parent cells. A significant increase in tubulin mRNA sequences is already evident 1 hr after the addition of Bt2cAMP to the neuroblastoma cells, and a maximal induction of 2-fold is seen after 12 hr. Continued treatment with Bt2cAMP for 4 days results in a down-regulation of the initial tubulin mRNA level independently of cell density. In the glioma cells Bt2cAMP also rapidly increases the level of tubulin mRNA sequences, reaching a maximum within 6 hr. However, in these cells the subsequent decrease in tubulin mRNA content depends on the culture's phase of growth: cells at the logarithmic growth phase do not down-regulate the tubulin mRNA content even after prolonged treatment with Bt2cAMP, whereas confluent cells do. The hybrid cell line manifests intermediate characteristics in Bt2cAMP regulation of tubulin mRNA level. The time course of induction and down-regulation of tubulin mRNA content observed in the hybrid cells is similar to that of the parent neuroblastoma, whereas the sensitivity to induction is glioma-like and is 8-fold over the initial level. Blot hybridization with labeled actin cDNA probes showed a similar but not identical induction of actin mRNA synthesis with the hybrid and glioma cells, whereas no significant change was observed with the neuroblastoma cells. Moreover, prolonged treatment with Bt2cAMP of all these cell lines did not result in down-regulation of actin mRNA sequences below the initial control value. It was also observed that the level of tubulin sequences in mRNA isolated from 12-day-old rat brain was higher than that in the newborn brain. However, at an age of 30 days, the level of tubulin sequences decreases to about 75% of the newborn level.
Protein-primed initiation of phage phi 29 DNA replication.
Authors: Watabe K , Shin M , Ito J
Published: May 2006

We recently reported the development of an in vitro replication system for bacteriophage phi 29 DNA. We have used this system for the isolation of replication activity associated with gene 3 protein (terminal protein) from phi 29-infected Bacillus subtilis cells. We utilized two assay systems: (i) DNA replication dependent on phi 29 DNA with the 5' end covalently linked to terminal protein (DNA-protein) and (ii) the formation of complex between the terminal protein and dAMP. The DNA-replication and the complex-forming activities were purified together through all steps. The complex of terminal protein and dAMP formed in the purified fraction was shown to serve as an effective primer for successive chain elongation in the presence of dNTPs by a pulse-chase experiment. The protein fraction purified from cells infected with a temperature-sensitive phi 29 mutant in gene 3 was thermolabile compared to the wild-type activity in the assay system for complex formation. This shows that the purified fraction having replication activity includes the gene 3 product of phi 29. Both the DNA replication and the complex formation activities are highly specific for phi 29 DNA-protein as template. The product analysis of elongated DNA revealed that the replication starts at both termini of the phi 29 genome. These results are consistent with the basic elements of the protein-priming model for the initiation of linear DNA synthesis.
Identification of homo-oligomers as potential intermediates in acetylcholine receptor subunit assembly.
Authors: Anderson D J , Blobel G
Published: May 2006

We have examined the sedimentation behavior, on sucrose density gradients, of acetylcholine receptor (AcChoR) subunits synthesized in vitro and integrated into heterologous rough microsomal membranes. In media containing nondenaturing detergents such as Triton X-100 or deoxycholate, the subunits appear to self-associate although, as previously reported, no heterologous interactions were detected. The sedimentation profiles assume a broad distribution in the region of 7-13 S. However, the peak fractions occupy the same region of the gradient as does native AcChoR, run in parallel. Such large homo-oligomers were not observed for another membrane protein, opsin, studied in the same way. This indicated that the associations are indeed between the AcChoR subunits and not simply between all newly synthesized membrane proteins. The homologous associations are interpreted to suggest a mechanism for maintaining the ionophore surfaces of the subunits in an energetically preferred, but metastable, configuration during the lengthy period of post-translational assembly.
Transmission of chloroplast genes in triploid and tetraploid zygospores of
Chlamydomonas reinhardtii
: Roles of mating-type gene dosage and gametic chloroplast DNA content
Authors: Matagne René F. , Mathieu Didier
Published: May 2006

Diploid clones homozygous (
mt
+
/
mt
+
or
mt
-
/
mt
-
) or heterozygous (
mt
+
/
mt
-
; phenotypically mt
-
) for the mating-type locus and homoplasmic for a chloroplast marker conferring resistance to an antibiotic were isolated by artificially induced cell fusion or sexual mating. These diploids were crossed with haploid or diploid strains of opposite mating type and carrying another chloroplast marker. The transmission of the chloroplast genes was analyzed in the triploid and tetraploid zygospores in comparison with diploid zygospores used as controls. The transmission was almost exclusively maternal (
mt
+
) (>94%) in the crosses
mt
+
×
mt
-
,
mt
+
/
mt
+
×
mt
-
, and
mt
+
/
mt
+
×
mt
-
/
mt
-
. The transmission was preferentially maternal (>76%) in the crosses
mt
+
×
mt
-
/
mt
-
whereas in the crosses
mt
+
×
mt
+
/
mt
-
, <50% of the zygospores transmitted the chloroplast allele of maternal (
mt
+
) origin. The zygospores produced in crosses
mt
+
/
mt
+
×
mt
+
/
mt
-
transmitted the alleles from both parents in >60% of cases. The results show that (
i
) the presence of one
mt
+
allele in the
mt
+
/
mt
-
(phenotypically mt
-
) diploid gametes and (
ii
) the higher amount of chloroplast DNA molecules (input) present in the diploid gametes versus the haploid ones favor the transmission of the chloroplast allele contributed by these gametes. Moreover, because the zygospores issued from crosses
mt
+
/
mt
+
×
mt
-
and
mt
+
×
mt
+
/
mt
-
were genotypically identical
mt
+
/
mt
+
/
mt
-
) but behaved very differently in their chloroplast gene transmission, it was concluded that the molecular events leading to preferential elimination of paternal DNA copies must occur before the fusion of nuclei or chloroplasts in the newly formed zygotes.
Amino acid sequence of the Fv region of a human monoclonal IgM (protein WEA) with antibody activity against 3,4-pyruvylated galactose in Klebsiella polysaccharides K30 and K33.
Authors: Goñi F , Frangione B
Published: May 2006

We have determined the amino acid sequence of the Fv [variable heavy (VH) and variable light (VL)] region of a human monoclonal IgM-kappa with antibody activity against 3,4-pyruvylated galactose, isolated from the plasma of patient WEA with Waldenström macroglobulinemia. The VH region has 114 residues, belongs to subgroup III, and has a very short third complementarity-determining region (CDR3), probably due to a small D segment/or an unusual D-J rearrangement (D, diversity; J, joining). The VL region has 108 residues and belongs to subgroup V kappa I. Compared to other members of the human VHIII and V kappa I families, WEA Fv does not appear to have significant differences within the framework residues but has unique CDRs that might be responsible for the particular antibody activity. Another IgM-kappa (GAL), which has an as-yet-undetermined antibody activity, shares a striking homology in V kappa with WEA, including an identical CDR1.
Anticodon shift in tRNA: a novel mechanism in missense and nonsense suppression.
Authors: Murgola E J , Prather N E , Mims B H , Pagel F T , Hijazi K A
Published: May 2006

In a previous publication, an unusual UGG-reading missense suppressor caused by insertion of an extra adenylate residue in the anticodon loop of an Escherichia coli glycine tRNA was described. In this study, we provide in vivo evidence that the additional nucleotide causes an "anticodon shift" by one nucleotide in the 3' direction and that the "new" anticodon can explain the unanticipated coding properties of the suppressor. We converted the UGG suppressor with ethyl methanesulfonate, a base-substitution mutagen, to suppressors that read codons related to UGG by a single base change. Sequence analysis of each mutant tRNA revealed that its mutational alteration was an anticipated base change in one of the three nucleotides of the "new" anticodon. Although the new suppressors read codons beginning with A or U, the mutant tRNAs lack the customary hypermodified nucleosides on the 3' side of the anticodon. As determined on the basis of their in vivo coding specificities, the new mutant tRNAs do not continue to utilize the original anticodon triplet for decoding. Furthermore, the failure of the UGG suppressor to correct frameshift mutations throughout each of three genes of the trp operon suggests that the addition of a nucleotide to the anticodon loop of a tRNA does not necessarily result in out-of-frame decoding by the tRNA. Therefore, a "frameshift" mutation in a tRNA has principally changed the triplet codon recognition properties of the molecule.
A conjugate decomposition of the Euclidean space
Authors: Han S.-P. , Mangasarian O. L.
Published: May 2006

Given a closed convex cone
K
in the
n
-dimensional real Euclidean space

R
n

and an
n
×
n
real matrix
A
that is positive definite on
K
, we show that each vector in

R
n

can be decomposed into a component that lies in
K
and another that lies in the conjugate cone induced by
A
and such that the two vectors are conjugate to each other with respect to
A
+

A
T

. As a consequence of this decomposition we establish the following characterization of positive definite matrices: An
n
×
n
real matrix
A
is positive definite if and only if it is positive definite on some closed convex cone
K
in

R
n

and (
A
+

A
T

)
-1
exists and is positive semidefinite on the polar cone
K
0
. If
K
is a subspace of

R
n

, then
K
0
is its orthogonal complement
K
[unk]. Other applications include local duality results for nonlinear programs and other characterizations of positive definite and semidefinite matrices.
Splicing of adenovirus RNA in a cell-free transcription system.
Authors: Padgett R A , Hardy S F , Sharp P A
Published: May 2006

A soluble whole-cell extract prepared accurately from HeLa cells splices 2-3% of the RNA transcribed from a DNA template containing the first and second leader exons of late adenovirus RNA. The spliced RNA was detected by a sensitive technique using hybridization to a single-stranded phage M13 cDNA clone, followed by binding to nitrocellulose filters. The identity of the spliced RNA was established by RNase T1 and pancreatic RNase two-dimensional peptide mapping. The bond formed during the in vitro splicing reaction appears to be a typical 3',5'-phosphodiester bond as judged by its sensitivity to RNase T1. The splicing reaction is specifically inhibited by KCl at concentrations greater than 50 mM and by the addition of cellular RNA. Three features of this system may account for the detection of splicing in a soluble extract: (i) the sensitive and unambiguous hybridization assay, (ii) the high transcriptional activity of the major late promoter of adenovirus, and (iii) the use of the first and second leader exon splice of adenovirus, which may be unusually rapid.
The leader mRNA of the histidine attenuator region resembles tRNAHis: possible general regulatory implications.
Authors: Ames B N , Tsang T H , Buck M , Christman M F
Published: May 2006

The leader region of the mRNA of the his operon is involved in regulating the frequency of transcription termination through attenuation and therefore expression of the his structural genes. We now report that the his leader mRNA has a remarkable sequence homology with the tRNAHis molecule. Of the 75 nucleotides forming tRNAHis (not counting the -CCA tail), 45 are homologous to nucleotide sequences found in the his leader mRNA. This homology extends to secondary structures which can form in the leader mRNA. The stems and loops of tRNAHis are thus related to those of the his leader mRNA which play a critical role in regulating expression of the his operon through attenuation. Many proteins that bind tRNAHis thus might bind to the similar structures found in the his leader mRNA and influence regulation by favoring the attenuator or anti-attenuator configuration. These include tRNA-modifying enzymes, the histidyl-tRNA synthetase, and the hisG enzyme. The significance of similar structures in other regulatory systems is discussed, particularly in relation to the role of tRNA-modifying enzymes as important regulatory molecules in both prokaryotes and eukaryotes.
Allosteric cofactor-mediated enzyme cooperativity: a theoretical treatment.
Authors: Kuo L C
Published: May 2006

The situation under which substrate cooperativity is apparent only in the presence of an inhibitor has been investigated. When a substrate and an inhibitor bind independently to a cooperative enzyme that conforms to the concerted Monod-Wyman-Changeux model, each of the two ligands must induce intersubunit transitions in the protein molecule in order to have their allosteric effects coupled to one another. The inhibitor exerts a heterotropic influence on the saturation function of the substrate and enhances the otherwise recondite homotropic effect of the latter. If the ligands bind competitively to the enzyme, however, intersubunit transitions in the enzyme need be induced only by the inhibitor. A sigmoidal substrate saturation curve is then obtained as a result of displacement of the inhibitor from the enzyme by the substrate. In this mechanism, the competitive inhibitor participates as a cofactor required for the expression of substrate cooperativity and the familiar ability of regulatory enzymes to mediate homotropic interactions directly between substrate molecules is absent. Experimental tests are proposed to elucidate the nature of cooperative interactions for enzymes that appear to retain heterotropic but not homotropic effects in substrate binding.
Epidermal growth factor and glucagon receptors in mice homozygous for a lethal chromosomal deletion.
Authors: Shaw P A , Gluecksohn-Waelsch S
Published: May 2006

The binding of epidermal growth factor (EGF) and of glucagon to their receptors has been examined in single-cell suspensions obtained from livers and other organs of newborn mice homozygous for a perinatally lethal deletion that includes the albino (c) locus on chromosome 7. Competition experiments with 125I-labeled and nonradioactive EGF and Scatchard analysis of equilibrium binding data showed that hepatocytes from deletion homozygotes had only approximately equal to 20% of the number of specific EGF receptors present in cells from normal littermates. In contrast, EGF binding to single-cell suspensions from organs other than the liver was normal in deletion homozygotes. Similar results were obtained in competitive displacement experiments with 125I-labeled and nonradioactive glucagon: hepatocytes from deletion mutants showed only approximately equal to 30% of the specific glucagon binding sites found in cells from normal littermates. As in the case of EGF, the decreased binding was due to decreased numbers of glucagon receptors per cell rather than alterations in receptor affinity, and glucagon binding to single-cell suspensions from organs other than the liver was normal in the deletion mutants. The reductions in numbers of EGF and glucagon receptors are liver-cell specific as are the previously described ultrastructural and biochemical abnormalities in these mutants. The significance of cell membrane integrity and hormone-receptor interactions in the control of normal liver cell differentiation is discussed.
Monoclonal antibody-directed effector cells selectively lyse human melanoma cells in vitro and in vivo.
Authors: Schulz G , Bumol T F , Reisfeld R A
Published: May 2006

Monoclonal antibody 9.2.27 (mAb 9.2.27) directed to a chondroitin sulfate proteoglycan on human melanoma cells was able to suppress tumor growth in athymic (nu/nu) mice more effectively when bound with polyethylene glycol to murine effector cells than when injected alone. These "armed" effector cells also proved more effective than the monoclonal antibody in eliciting antibody-dependent cellular cytotoxicity against human melanoma target cells in vitro.
Alterations in Ca2+ binding by and composition of the cardiac sarcolemmal membrane in chronic diabetes.
Authors: Pierce G N , Kutryk M J , Dhalla N S
Published: May 2006

Chronic streptozotocin-induced diabetes in rats was associated with a significant loss in the ability of isolated cardiac sarcolemmal membranes to bind Ca2+. Administration of insulin to the diabetic rats normalized the sarcolemmal Ca2+ binding capacity. The content of sialic acid residues, which are considered to represent a superficial Ca2+ pool in sarcolemma, was decreased in preparations from diabetic rats, and this change also was reversible upon insulin treatment of the diabetic rats. Treatment of sarcolemma with neuraminidase decreased Ca2+ binding by 37% in control preparations but had no effect on diabetic preparations. Diphosphatidylglycerol content was decreased but other acidic phospholipids such as phosphatidylinositol and phosphatidylserine, which also bind Ca2+, were not altered during diabetes. An increase in lysophosphatidylcholine and a decrease in phosphatidylethanolamine contents were observed in membranes isolated from diabetic rats. These results suggest that some alterations occur in Ca2+ binding and composition of heart sarcolemma in chronically diabetic rats and may provide further insight into the pathogenesis of diabetic cardiomyopathy.
Metabolism of leukotriene A4 by an enzyme in blood plasma: a possible leukotactic mechanism.
Authors: Fitzpatrick F , Haeggström J , Granström E , Samuelsson B
Published: May 2006

Cell-free mammalian plasma transformed leukotriene A4 into leukotriene B4. This conversion originated from a soluble enzymatic activity. Heating at 56 degrees C or digestion of plasma with a proteolytic enzyme eliminated formation of leukotriene B4 but not other diastereomeric dihydroxyicosatetraenoic acids formed by nonenzymatic hydrolysis of leukotriene A4. Plasma from several mammals, including guinea pigs, pigs, cows, sheep, rabbits, rats, dogs, and humans, exhibited a qualitatively similar activity. Maximal production of leukotriene B4 occurred in guinea pig plasma near a pH of 7.6; metabolic capacity approached saturation at a substrate concentration of 10 microM. Leukotriene A4 anion was transformed but not its methyl ester. The results suggest that conversion of leukotriene A4 by an enzyme in blood plasma of mammals may provide local gradients of the chemotactic substance leukotriene B4. High interfacial concentrations of leukotriene B4 between the vascular endothelium and leukocytes could facilitate their diapedesis and accumulation at inflammatory sites.
Heparin-catalyzed inhibitor/protease reactions: kinetic evidence for a common mechanism of action of heparin.
Authors: Griffith M J
Published: May 2006

Three different heparin-catalyzed inhibitor/protease reactions were studied: antithrombin III/thrombin, heparin cofactor II/thrombin, antithrombin III/factor Xa. The three reactions were saturable with respect to both inhibitor and protease. The initial reaction velocity, for each reaction, could be described by the general rate equation for a random-order bireactant enzyme-catalyzed reaction. The kinetic parameters for the heparin-catalyzed antithrombin III/thrombin and antithrombin III/factor Xa reactions differed in terms of apparent maximum velocity (Vmax) and apparent heparin-protease dissociation constant values. The apparent heparin-antithrombin III dissociation constant values were the same for both reactions. The kinetic parameters for the heparin-catalyzed antithrombin III/thrombin and heparin cofactor II/thrombin reactions differed in terms of apparent Vmax and apparent heparin-inhibitor dissociation constant values. The apparent heparin-thrombin dissociation constant values were the same for both reactions. The results are consistent with a general mechanism of action of heparin for the three reactions that, in its simplest form, requires only that both protease and inhibitor bind to heparin for catalysis to occur.
Primary structure of streptococcal Pep M5 protein: Absence of extensive sequence repeats
Authors: Manjula Belur N. , Mische Sheenah M. , Fischetti Vincent A.
Published: May 2006

Extensive sequence repeats have been observed in a biologically active fragment of type 24 streptococcal M protein, namely Pep M24 [Beachey, E. H., Sayer, J. M. & Kang, A. H. (1978)
Proc. Natl. Acad. Sci. USA
75, 3163-3167]. To determine whether such extensive repetition in sequence is a common characteristic of the antiphagocytic streptococcal M proteins, we have determined the sequences of the clostripain peptides of Pep M5, a biologically active fragment of the type 5 M protein that is analogous to Pep M24. These sequences, together with the amino-terminal sequence of the whole molecule, accounted for nearly two thirds of the Pep M5 molecule. However, extensive identical repeats of the kind observed in Pep M24 were not present in Pep M5. Preliminary study of the amino acid sequence analysis of the M protein from type 6 Streptococcus has also indicated the absence of sequence repeats within the regions of this molecule examined so far. These results suggest that extensive sequence repeats may not be a common characteristic of M-protein molecules. On the other hand, the seven-residue periodicity of the nonpolar residues, a characteristic of α-helical coiled-coil structures, appeared to extend over most of the Pep M5 molecule. This feature has been observed previously for the partial sequences of three M protein serotypes. Thus, the important element of the M-protein structure appears to be the seven-residue periodicity necessary for the maintenance of the coiled-coil structure rather than extensive identical amino acid sequence repeats.
Identification of a single transcriptional initiation site for the glutamic tRNA and COB genes in yeast mitochondria.
Authors: Christianson T , Edwards J C , Mueller D M , Rabinowitz M
Published: May 2006

We have identified a single transcriptional initiation site for the glutamic tRNA and COB (cytochrome b) genes by using the complementary techniques of in vitro capping of RNA and in vitro transcription. In the capping reaction, mitochondrial RNA is labeled with [alpha-32P]GTP by vaccinia virus guanylyltransferase. This reaction is specific for the 5' ends of RNA retaining the terminal triphosphate of transcriptional initiation. Exploiting the extremely low G+C content (18%) of yeast mitochondrial DNA, we digested in vitro capped transcripts from various petite deletion mutants with the G-specific RNase T1. By petite deletion mapping, a capped transcript giving rise to a 51-base RNase T1-generated oligonucleotide was localized near the glutamic tRNA gene. When the sequence of this oligonucleotide was determined, it perfectly matched the DNA sequence 391 base upstream of the glutamic tRNA. Purified yeast mitochondrial RNA polymerase initiated transcription in vitro at the same site as shown by the sequence of the 33-base oligonucleotide product of the reaction performed in the absence of CTP. Initiation starts at a nonanucleotide sequence previously implicated in yeast mitochondrial transcriptional initiation. Because there is no evidence of an initiation site in the 1,050 bases between the glutamic tRNA and COB genes, the two genes are likely to be transcribed together. Further evidence of a long common transcript was provided by RNA blot hybridization.
Macromolecular crowding allows blunt-end ligation by DNA ligases from rat liver or Escherichia coli.
Authors: Zimmerman S B , Pheiffer B H
Published: May 2006

In the presence of high concentrations of any of several types of macromolecules, DNA ligase preparations from rat liver nuclei or from Escherichia coli actively catalyze the blunt-end ligation of DNA. This is in contrast to the lack of activity on such substrates by these enzymes under conventional assay conditions. In addition, the previously established activity of T4 DNA ligase on blunt-ended molecules is greatly increased in the presence of high concentrations of macromolecules. Because such crowded solutions may well be a more adequate model for intracellular conditions than assays in dilute solutions, we suggest that blunt-end ligation may be a widely occurring reaction in vivo.
Bacillus stearothermophilus contains a plasmid-borne gene for alpha-amylase.
Authors: Mielenz J R
Published: May 2006

The gene for thermostable alpha-amylase from the thermophilic bacterium Bacillus stearothermophilus has been cloned and expressed in Escherichia coli. Each alpha-amylase-producing colony contained at least a 9.7-kilobase-pair (kb) chimeric plasmid composed of the vector pBR322 and a common 5.4-kb HindIII fragment of DNA. B. stearothermophilus contains four plasmids with sizes from 12 kb to over 108 kb. Restriction endonuclease analysis of these naturally occurring plasmids showed they also contain a 5.4-kb HindIII fragment of DNA. Cloning experiments with the four plasmids yielded alpha-amylase-producing E. coli that contained the same 9.7-kb chimeric plasmid. Restriction endonuclease analysis and further recombinant DNA experiments identified a 26-kb plasmid that contains the gene for alpha-amylase. A spontaneous mutant of B. stearothermophilus unable to produce alpha-amylase was missing the 26-kb plasmid but contained a 20-kb plasmid. A 6-kb deletion within the region of the 5.4-kb HindIII fragment yielded the 20-kb plasmid unable to code for alpha-amylase. A nick-translated probe for the alpha-amylase coding region did not hybridize to either plasmid or total cellular DNA from this mutant strain of B. stearothermophilus. These results demonstrate the gene for alpha-amylase is located exclusively on a 26-kb plasmid in B. stearothermophilus with no genetic counterpart present on the chromosome.
A canine model of human alpha-L-iduronidase deficiency.
Authors: Spellacy E , Shull R M , Constantopoulos G , Neufeld E F
Published: May 2006

A disease discovered in three Plott Hound littermates was found to be associated with a profound and specific deficiency of alpha-L-iduronidase (mucopolysaccharide alpha-L-iduronohydrolase; EC 3.2.1.76) in fibroblasts and leukocytes. The pedigree was consistent with autosomal recessive inheritance. A markedly increased amount of dermatan sulfate and heparan sulfate was excreted in urine. Fibroblasts cultured from the skin of the affected dogs accumulated excessive 35S-labeled mucopolysaccharide; this accumulation could be decreased to a normal level by exogenous human high-uptake alpha-L-iduronidase (Hurler corrective factor) as well as by secretions of normal human or canine fibroblasts. The correction was inhibited by mannose 6-phosphate. Maturation of alpha-L-iduronidase in normal canine fibroblasts followed the pathway previously observed in human fibroblasts; no cross-reactive material was observed in the cells or in secretions from the fibroblasts of the affected dogs. The canine disorder thus resembles mucopolysaccharidosis I in all biochemical parameters tested; the clinical appearance of the animals is closest to Hurler-Scheie syndrome, a form of alpha-L-iduronidase deficiency of intermediate severity. The animal model should prove valuable for therapeutic experiments.
Autoradiographic localization of a binding protein(s) specific for prostaglandin D2 in rat brain.
Authors: Yamashita A , Watanabe Y , Hayaishi O
Published: May 2006

The specific [3H]prostaglandin (PG) D2 binding was detected by using the slide-mounted sections of rat brain fixed by perfusion with 2% paraformaldehyde. The binding was reversible, saturable, high affinity, Na+ dependent, and highly specific for PGD2. These binding characteristics are essentially similar to those observed with the synaptic membrane of rat brain as previously reported. Using autoradiographic image analyses by computerized densitometry and color coding, we visualized the localization of [3H]PGD2 binding in rat brain. A high density of the binding sites was observed in the cerebral cortex, preoptic area, amygdala, hypothalamic nuclei (arcuate nucleus, ventromedial nucleus, and posterior hypothalamic nucleus), thalamic nuclei (reuniens nucleus and rhomboid nucleus), hippocampus, pineal body, and cerebellar cortex. The binding was not significantly observed in the striatum and also was negative in the white matter, arachnoid membranes, and vasculatures.
On the state of aggregation of newly secreted procollagen.
Authors: Hulmes D J , Bruns R R , Gross J
Published: May 2006

Procollagen and partially processed procollagen from cultures of primary chicken embryo tendon cells appeared as segment-long-spacing (SLS)-like aggregates when drops of medium were negatively stained and examined by electron microscopy. Similar aggregates were obtained after negative staining of medium partially purified by gel filtration and also after staining thin sections of fixed, dehydrated, and embedded pellets formed by prolonged ultracentrifugation of whole culture medium. In contrast to results from electron microscopy, analysis by velocity density gradient sedimentation or sedimentation equilibrium indicated the exclusive presence of procollagen or partially processed procollagen monomers in solution. These contradictory data can be reconciled if procollagen exists in monomeric form when greatly diluted (as in culture medium), and in specific aggregated form (SLS) at high concentration. We believe that cells in vivo secrete procollagen in high, local concentration packaged in the SLS form. We propose that such zero-D arrayed packages are the precursors of native collagen fibrils.
Autoimmune damage to spermatogenesis in rodents immunized with mouse F9 embryonic carcinoma cells.
Authors: Vojtiskova M , Pokorna Z , Draber P
Published: May 2006

Significant inhibition of spermatogenesis and appearance of antibodies against spermatogenic cells identified by cytotoxicity and immunofluorescence reactions were observed in mice of inbred strains 129/Sv and BALB/c and in albino guinea pigs after syngeneic, allogeneic, and xenogeneic immunization with mouse F9 embryonic carcinoma cells and Freund's complete adjuvant. A similar syngeneic immunization with PYS-2 cells was ineffective. Appropriate absorption experiments confirmed the similarity between the antigens of F9 and spermatogenic cells and the absence of such a similarity with antigens of PYS-2 cells. These results support the hypothesis that the oncofetal F9 antigens represent spermatogenic differentiation antigens and thus play an essential role in spermatogenic cell differentiation.
Specific in vitro binding of a plasmid to a membrane fraction of Bacillus subtilis.
Authors: Korn R , Winston S , Tanaka T , Sueoka N
Published: May 2006

A model system has been developed to study the in vitro binding of a plasmid to the membrane fraction from Bacillus subtilis. The plasmid DNA molecule used in these studies was pSL103 (8.0 kilobases), a chimeric plasmid consisting of a Staphylococcus aureus plasmid (pUB110, 4.5 kilobases) and a DNA fragment (3.5 kilobases) from Bacillus pumilus carrying trpC+ gene. This plasmid replicates in B. subtilis cells, and its in vivo membrane binding (as well as its replication) is dependent on the product of a DNA initiation gene, dna-1, of B. subtilis. In this paper we demonstrate the in vitro specific binding of exogenous pSL103 to the isolated membrane fraction. This in vitro binding is specific to the origin-containing portion (pUB110) of pSL103. The trpC+-carrying portion neither binds to the membrane fraction nor competes with pSL103 for binding to the membrane fraction in vitro. ColE1 plasmid, which does not replicate in B. subtilis, neither binds to the B. subtilis membrane fraction nor competes with pSL103 for binding.
Species-specific gating of gametangial dehiscence as a temporal reproductive isolating mechanism in
Coelomomyces
Authors: Federici Brian A.
Published: May 2006

Hybridzygotes of
Coelomomyces dodgei
and
Coelomomyces punctatus
can be produced in the laboratory by making reciprocal crosses of their gametes. Although the F
1
hybrid sporophyte is viable, the F
1
gametophyte is inviable. Gametangial dehiscence (gamete release) in both species is relatively synchronous and follows a photoperiod-dependent diel periodicity, but even in populations grown under identical conditions, the gametes of each species are released during significantly different time frames. When the fungi are reared on an 8/16-hr dark/light regime, the gametes of
C. punctatus
are released during hours 7-11, whereas those of
C. dodgei
are released during hours 16-19. These results demonstrate that gametangial dehiscence is gated and, furthermore, indicate that this gating serves as a temporal reproductive isolating mechanism for these species of
Coelomomyces
. Additional preliminary studies with
C. dodgei
demonstrate that gametogenesis and gametangial dehiscence are photoperiod-dependent functions triggered by the onset of the dark period during the last day of gametophyte development, a dark period as short as 2 hr being sufficient to stimulate gametogenesis and gametangial dehiscence.
Energetics of B-to-Z transition in DNA.
Authors: Peck L J , Wang J C
Published: May 2006

Analysis by two-dimensional gel electrophoresis of topoisomers of plasmids containing d(pCpG)n . d(pCpG)n inserts, in which n ranges between 8 and 21, shows that the B-to-Z transition within the alternating C-G is readily induced by negative supercoiling and is highly cooperative. The free energy parameters for the transition in dilute aqueous buffers have been evaluated from a statistical mechanical analysis of the data, and these parameters allow prediction of the superhelicities of plasmids at which the transition occurs in alternating C-G inserts over a wide range of lengths. In agreement with the crystal structures, the helical handedness of the B structure in solution and that of the Z structure are shown to be opposite to each other. Furthermore, it is found that the B form of the alternating C-G sequence in solution has a helical periodicity of 10.5 +/- 0.1 base pairs per turn, and the Z form has a helical periodicity of 11.6 +/- 0.3 base pairs per turn. There also appears to be a significant unwinding of the right-handed DNA duplex at each of the B/Z junctions.
The chorionic gonadotropin alpha-subunit gene is on human chromosome 18 in JEG cells.
Authors: Hardin J W , Riser M E , Trent J M , Kohler P O
Published: May 2006

The gene for the alpha subunit of human chorionic gonadotropin (hCG) has been tentatively assigned to human chromosome 18. This localization was accomplished through the use of Southern blot analysis. A full-length cDNA probe for the hCG alpha subunit and DNA isolated from a series of somatic hybrids between mouse and human cells were utilized to make this assignment. In addition, in situ hybridization with normal human peripheral blood lymphocytes as a source of human chromosomes and with the same cDNA probe confirmed this result. The presence of human chromosome 18 was required for the detection of DNA fragments characteristic of the alpha-hCG gene. These results are consistent with our previous observation that human chromosomes 10 and 18 are required for the production of hCG in cultured cells.
Intracistronic complementation in the simian virus 40 A gene.
Authors: Tornow J , Cole C N
Published: May 2006

A set of eight simian virus 40 mutants was constructed with lesions in the A gene, which encodes the large tumor (T) antigen. These mutants have small deletions (3-20 base pairs) at either 0.497, 0.288, or 0.243 map units. Mutants having both in-phase and frameshift mutations at each site were isolated. Neither plaque formation nor replication of the mutant DNAs could be detected after transfection of monkey kidney cells. Another nonviable mutant, dlA2459, had a 14-base-pair deletion at 0.193 map unit and was positive for viral DNA replication. Each of the eight mutants were tested for ability to form plaques after cotransfection with dlA2459 DNA. The four mutants that had in-phase deletions were able to complement dlA2459. The other four, which had frameshift deletions, did not. No plaques were formed after cotransfection of cells with any other pair of group A mutants. This suggests that the defect in dlA2459 defines a distinct functional domain of simian virus 40 T antigen.
Synthesis and assembly of human small nuclear ribonucleoproteins generated by cell-free translation.
Authors: Fisher D E , Conner G E , Reeves W H , Blobel G , Kunkel H G
Published: May 2006

Cell-free translation of human poly(A)+ RNA was carried out to generate and analyze the protein constituents of small nuclear ribonucleoprotein (snRNP) particles. The snRNP proteins were identified by immunoprecipitation with sera from patients with systemic lupus erythematosus. Size fractionation of mRNA prior to translation revealed that these snBNP proteins are all encoded by separate messages. One of the proteins (the A protein, molecular weight 32,000) was seen to lose antigenicity upon RNase treatment either when extracted from cells or when generated in vitro. RNase treatment of immunoprecipitated snRNPs released the A protein in an electrophoretically pure form. Analysis of snRNPs translated in vitro revealed the presence of unassembled and assembled particles as determined by sucrose density gradient sedimentation. Post-translational assembly of snRNPs involving both RNA-protein binding (as revealed by A protein antigenicity) and associations of other snRNP proteins occurred in the in vitro system employed here. In addition, the presence of unassembled snRNP proteins permitted the determination of the precise antigen peptides recognized by Sm and RNP autoimmune sera. It was observed that Sm sera are capable of recognizing each of the eight snRNP proteins, whereas RNP sera recognize only two of the eight.
Correction of a genetically caused enzyme defect by somatic cell hybridization.
Authors: Cori C F , Gluecksohn-Waelsch S , Shaw P A , Robinson C
Published: May 2006

Liver cells obtained from newborn mice homozygous for any one of several overlapping deletions in chromosome 7 fail to express a number of liver-specific differentiated traits. Among these is the activity of the membrane-bound liver-specific enzyme glucose-6-phosphatase (Glc-6-Pase; D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9). Previous studies have led to the suggestion that the region of the genome covered by these deletions includes genes that normally regulate the expression of structural genes encoding liver-specific enzymes and proteins mapping elsewhere in the genome. To find out whether the deficiency of Glc-6-Pase may be caused by the deletion of the relevant structural gene, mouse liver cells homozygous for the deletion c14CoS were hybridized with 2S Faza rat hepatoma cells, and the hybrid cell cultures were analyzed for mouse and rat Glc-6-Pase activity. Hybrids showed expression of mouse Glc-6-Pase activity, proving that the structural gene for this enzyme is not included in the deletion c14CoS in chromosome 7. In the hybrid cells the rat hepatoma genome apparently contributes a factor that activates the structural gene of the mouse and corrects its failure of expression, which most likely resulted from the deletion of an essential regulatory or processing gene. By using as a marker glucose-6-phosphate isomerase (Glc-6-PIase; glucosephosphate isomerase, D-glucose-6-phosphate ketolisomerase, EC 5.3.1.9), known to map on chromosome 7, this entire chromosome could be excluded as a possible carrier of the Glc-6-Pase structural gene. In addition, the structural genes for Glc-6-Pase and for tyrosine aminotransferase (TyrATase; L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5), another enzyme deficient in lethal deletion homozygotes, were shown to map on two different chromosomes. Together with our previous studies of TyrATase gene regulation, the present experiments suggest that the region of the mouse genome defined by the deletions includes one or more genes regulating the expression of several structural genes that map on different chromosomes and that encode liver-cell-type specific traits.
A lymphocyte antigen encoded by a locus closely linked to H-3 on the second chromosome of the mouse.
Authors: Gasser D L , Ziebur J , Matsumoto K
Published: May 2006

A monoclonal antibody has been developed that recognizes a mouse lymphocyte antigen, Ly-23, that is controlled by an H-3 histocompatibility complex-linked gene on the second chromosome. Competitive binding experiments demonstrate that this antigen is distinct from the polymorphic beta 2-microglobulin antigen, which also is encoded by a gene closely linked to H-3. The strain distribution pattern differs from that of the beta 2-microglobulin polymorphism, and recombination apparently has separated the genes for these two antigens during the derivation of one recombinant inbred line. However, the R1E/TL8X line of cells, which lacks beta 2-microglobulin, is negative for the Ly-23 antigen, although the control R1.1 line is positive. The H-2 locus affects the expression of both the Ly-23 and beta 2-microglobulin antigens.
Activation of antigen-specific B cells: role of T cells, cytokines, and antigen in induction of growth and differentiation.
Authors: Noelle R J , Snow E C , Uhr J W , Vitetta E S
Published: May 2006

T cells and cytokines were used to activate highly enriched populations of 2,4,6-trinitrophenyl (TNP)-binding B cells (TNP-ABC). TNP-ABC did not proliferate or differentiate when they were cultured with thymus-dependent (TD) antigen, even in the presence of supernatants known to contain B-cell growth and differentiation factors. However, purified TNP-ABC did proliferate and differentiate when they were cultured with TD antigen in the presence of carrier-primed T cells and antigen (TNP-keyhole limpet hemocyanin)--i.e., linked recognition. TNP-ABC blasts generated under conditions of linked recognition proliferated and differentiated in response to cytokines in the absence of T cells and antigen. In contrast, under conditions of nonlinked recognition (hapten and carrier on different molecules) TNP-ABC blasts also proliferated but did not differentiate in response to the same cytokines. These results indicate that antigen-specific "resting" B cells must be activated by T cells and antigen prior to becoming responsive to cytokines. Furthermore, activation under conditions of linked and nonlinked recognition generates two different types of blasts with regard to their subsequent response to cytokines.
Identification by transfection of transforming sequences in DNA of human T-cell leukemias.
Authors: Souyri M , Fleissner E
Published: May 2006

DNA from human T-cell leukemia cell lines was tested for focus-inducing activity on cultures of NIH 3T3 cells. Three leukemias yielded DNA active in this assay; restriction enzyme sensitivity of this activity indicated that similar, relatively large DNA sequences were involved. Southern blot analysis revealed conserved size classes of restriction fragments containing human repetitive (Alu) sequences in serially transfected foci derived from the active DNAs. Similar blot hybridizations with a probe specific for the human N-ras oncogene detected a 9-kilobase EcoRI fragment in all cases. DNA containing this fragment from one of the leukemias, molecularly cloned in bacteriophage lambda, displayed highly amplified focus-inducing activity in transfection assays. Thus, the N-ras oncogene appears to be active in these three human leukemias of T-cell origin.