bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of the polypeptides encoded in the ATPase 6 gene and in the unassigned reading frames 1 and 3 of human mtDNA.
Authors: Chomyn A , Mariottini P , Gonzalez-Cadavid N , Attardi G , Strong D D , Trovato D , Riley M , Doolittle R F
Published: May 2006

Antibodies prepared against chemically synthesized peptides predicted from the DNA sequence have been used to identify the polypeptides encoded in the ATPase 6 gene and in unidentified reading frames (URFs) 1 and 3 of human mtDNA. In particular, antibodies directed against the COOH-terminal nonapeptide of the putative polypeptide encoded in the ATPase 6 reading frame immunoprecipitated specifically component 17 of the HeLa cell mitochondrial translation products, the reaction being inhibited by the specific peptide. Similarly, antibodies directed against the COOH-terminal undecapeptide of the putative URF1 product or against the COOH-terminal heptapeptide of the presumptive URF3 product were effective in immunoprecipitating specifically component 12 or, respectively, component 24 of the mitochondrial translation products. The sizes of proteins 17, 12, and 24, as estimated from their electrophoretic mobilities, are compatible with their being the products of the ATPase 6 gene, URF1, and URF3, respectively.
Two DNA methyltransferases from murine erythroleukemia cells: purification, sequence specificity, and mode of interaction with DNA.
Authors: Bestor T H , Ingram V M
Published: May 2006

Dye-ligand chromatography on Cibacron blue F3GA-agarose has been used to resolve two species of DNA (cytosine-5-)-methyltransferase from nuclear extracts of uninduced Friend murine erythroleukemia cells. Each species has been highly purified; the activities in the first and second peaks were associated with polypeptides of Mr 150,000 and 175,000, respectively. Analysis of substrate specificity with synthetic DNAs and restriction fragments of phi X174 replicative form DNA and pBR322 DNA showed that neither enzyme had dependence on the sequence context of CpG dinucleotides; poly(dG-dC) had the greatest methyl-accepting activity of any unmethylated DNA substrate tested. De novo methylation by both enzymes was inefficient relative to methylation of hemimethylated sites. Methyl-accepting activity was strongly dependent on DNA chain length. This observation suggests that binding to DNA, followed by one-dimensional diffusion of enzyme along the DNA molecule, is important in the mechanism by which DNA methyltransferase locates its recognition sites.
Formation of receptosomes from plasma membrane coated pits during endocytosis: analysis by serial sections with improved membrane labeling and preservation techniques.
Authors: Willingham M C , Pastan I
Published: May 2006

The early events in the entry of ligands into cultured fibroblasts through plasma membrane clathrin-coated pits were examined by using serial sections and electron microscopy. Swiss 3T3 cells were labeled at 4 degrees C with concanavalin A-horseradish peroxidase and warmed to 37 degrees C for 1 or 2 min to label organelles participating in endocytosis. Cells were fixed in glutaraldehyde and further preserved with a membrane contrast-enhancement technique that utilized sequential osmium/ferrocyanide, thiocarbohydrazide, and osmium/ferrocyanide treatment in situ. This technique provides exceptional preservation and enhanced contrast of membranous structures. Serial sections taken both parallel to and perpendicular to the substratum were examined by electron microscopy, and 263 clathrin-coated structures containing horseradish peroxidase were evaluated for their connection to the plasma membrane. All of these coated structures were found to be connected to the plasma membrane either directly or via necks approximately equal to 170 A in width and up to 7,000 A in length. These necks were often surrounded by a dense filamentous network. These results strongly suggest that isolated coated vesicles do not form during the endocytosis process in Swiss 3T3 cells.
Early differential tissue expression of transposon-like repetitive DNA sequences of the mouse.
Authors: Brûlet P , Kaghad M , Xu Y S , Croissant O , Jacob F
Published: May 2006

Another family of long moderately repetitive and dispersed sequences has been identified in the mouse genome. These sequences have a transposon-like structure. A 6-kilobase RNA transcript is detected in undifferentiated embryonal carcinoma cell lines but not in any of the differentiated cell types tested. By R-loop formation, the RNA is colinear with a DNA fragment from a randomly selected genomic clone.
Pre-crisis mouse cells show strain-specific covariation in the amount of 54-kilodalton phosphoprotein and in susceptibility to transformation by simian virus 40.
Authors: Chen S , Blanck G , Pollack R E
Published: May 2006

We have used several inbred mouse strains to examine the role of the 54-kilodalton (kDa) cellular phosphoprotein in transformation by the papovavirus simian virus 40. We have measured the endogenous 54-kDa phosphoprotein in cells obtained from these inbred mouse strains. To study the effect of passage, cell cultures were measured for amount of the 54-kDa phosphoprotein at the 2nd and 12th passages. In the absence of any transforming agent, the amount of endogenous 54-kDa phosphoprotein in early pre-crisis mouse cells varied in a strain-specific way. Transformation frequency varied coordinately with endogenous 54-kDa expression. Mouse strains whose cells produced a high level of endogenous 54-kDa phosphoprotein on passage did not further increase its expression after simian virus 40 transformation.
Evidence for a proenkephalin-like precursor in amphibian brain.
Authors: Kilpatrick D L , Howells R D , Lahm H W , Udenfriend S
Published: May 2006

The mammalian proenkephalin-derived peptides [Leu]enkephalin, [Met]enkephalin, and [Met]enkephalin-Arg6-Phe7 were identified in acid extracts of the brain of Bufo marinus by using reversed-phase HPLC and specific radioimmunoassays. [Met]Enkephalin was the predominant opioid peptide present (270 pmol/g). In contrast, the octapeptide [Met]enkephalin-Arg6-Gly7-Leu8, which is also derived from mammalian proenkephalin, was not detected. The ratio of free [Met]enkephalin to [Met]enkephalin-Arg6-Phe7 was found to be 3.5 to 1, which is similar to that observed in mammalian proenkephalin-containing tissues. Together these data (i) indicate that amphibian brain contains a proenkephalin related to the mammalian precursor and (ii) establish the existence of enkephalins and proenkephalin-derived enkephalin-containing peptides in a submammalian species.
A small region of the mouse mammary tumor virus long terminal repeat confers glucocorticoid hormone regulation on a linked heterologous gene.
Authors: Majors J , Varmus H E
Published: May 2006

Expression of mouse mammary tumor virus (MMTV) proviruses is transcriptionally regulated by glucocorticoid hormones. We have linked the MMTV long terminal repeat (LTR) to the coding region of the herpes simplex virus thymidine kinase gene and used this construction to characterize sequences within the LTR that are involved in glucocorticoid regulation. Our results show that 290 base pairs (bp) of the MMTV LTR, including 190 bp upstream from the start site for transcription, are sufficient to confer regulation on the downstream gene. Deletion of an additional 50 bp, leaving sequences from position -140 to +100, eliminates the response. However, the constitutive level of expression is maintained even after deletion of sequences upstream from position -80, indicating that sequences required for the hormone response can be distinguished from those required for basal expression. We also have shown, by making a 4-bp insertion or a 20-bp deletion around position -107, that the distance between the putative signal for hormone response and the start site of transcription can be varied without affecting regulation. Furthermore, replacement of MMTV sequences from position -59 to +100 with analogous sequences from the Rous sarcoma virus LTR does not change the regulation.
Identification and characterization of human haptoglobin cDNA.
Authors: Yang F , Brune J L , Baldwin W D , Barnett D R , Bowman B H
Published: May 2006

Recombinant plasmids containing human cDNA encoding haptoglobin, a plasma protein that binds free hemoglobin, have been isolated by screening an adult human liver library with a mixed oligonucleotide probe. Four cDNA clones containing inserts have been obtained that span 1,218 nucleotides of the haptoglobin coding sequence, including the 3' end of the haptoglobin cDNA. The cDNA sequence included a leader sequence followed by alpha 2-chain and beta-chain sequences. A heretofore unseen arginine residue was deduced between the human alpha- and beta-chain sequences. This is a probable site of limited proteolysis leading to the formation of the alpha and beta polypeptides in mature haptoglobin. A comparison of the haptoglobin alpha-beta-junction region and the heavy-light-chain junction of tissue-type plasminogen activator strengthens the evolutionary homology found in haptoglobin and the serine proteases. The Hp alpha 2 gene, which was shown earlier to be a partial duplication produced by unequal crossing-over between Hp alpha 1 genes, has been impossible to align by protein characterization. The cDNA sequence establishes the alignment of Hp alpha 2FS in the Hp alpha 2 gene studied here.
Human ferritin gene is assigned to chromosome 19.
Authors: Caskey J H , Jones C , Miller Y E , Seligman P A
Published: May 2006

Ferritin is the intracellular iron storage protein. Tissue ferritin stores are markedly increased in hemochromatosis, a disease of iron overload that has been linked to chromosome 6. In order to provide further information concerning the genetics of ferritin synthesis and to determine if the structural gene for ferritin was on chromosome 6, studies were performed to identify the human chromosome that contains the ferritin gene. Ferritin immunoassays were performed on extracts of Chinese hamster ovary somatic cells that were hybridized with human lymphocytes and fibroblasts and contained various human chromosomes in different combinations. None of the 13 cell lines that lacked immunoreactive human ferritin contained chromosome 19, and all 9 of the cell lines that produced human ferritin contained chromosome 19. No other human chromosome shared this association with human ferritin. In studies of subclones of ferritin-positive cell lines, immunoreactive ferritin consistently segregated only with chromosome 19. Immunoprecipitation studies performed on cells that had been incubated with 59Fe-containing transferrin indicated that chromosome 19-containing cells incorporated iron into intact and functional molecules of human ferritin. The necessary and exclusive association of chromosome 19 with human ferritin indicates that a defect in the structural gene for human ferritin cannot account for the abnormalities of hemochromatosis. Moreover, this hamster-human hybrid cell system should prove useful in further studies of regulation of ferritin concentration and composition.
Mutations in PEP4 locus of Saccharomyces cerevisiae block final step in maturation of two vacuolar hydrolases.
Authors: Zubenko G S , Park F J , Jones E W
Published: May 2006

The biosynthesis of carboxypeptidase Y (EC 3.4.16.1) and proteinase A (EC 3.4.23.6) in yeast cells involves a series of posttranslational events, the last of which is dependent upon a function supplied by the PEP4 gene. Because pep4 mutations result in a 90-95% reduction in the levels of activity of at least three additional vacuolar hydrolases, it is likely that these, and perhaps all, yeast vacuolar hydrolases are synthesized as inactive precursors, which mature by a common mechanism that depends upon a function supplied by the PEP4 gene. The pep4 mutation shows an apparent gene dosage effect on levels of activity of proteinases A and B but not on the level of activity of carboxypeptidase Y. This effect appears to come about because the maturation machinery is capable of discriminating among these hydrolase precursors.
Alternative forms of lethality in mitomycin C-induced bacteria carrying ColE1 plasmids
Authors: Suit Joan L. , Fan M.-L. Judy , Sabik Joseph F. , Labarre Robert , Luria S. E.
Published: May 2006

We have studied the physiological effects of mitomycin C induction on cells carrying ColE1 plasmids with differing configurations of three genes: the structural gene coding for colicin (
cea
), a gene responsible for mitomycin C lethality (
kil
) that we located as part of an operon with
cea
, and the immunity (
imm
) gene, which lies near
cea
but is not in the same operon.
kil
is close to or overlaps
imm
. When
cea
+
plasmids are present mitomycin C induction results in 100-fold or greater increases in the level of colicin. Within an hour after induction more than 90% of cells carrying
cea
+
kil
+
plasmids are killed and macromolecular synthesis stops, capacity for transport of proline, thiomethyl β-D-galactoside, and α-methyl glucoside is lost, and the membrane becomes abnormally permeable as indicated by an increased accessibility of intracellular β-galactosidase to the substrate
o
-nitrophenyl β-D-galactoside. All of these events occur when a
cea
-
kil
+
imm
+
plasmid is present and none does when the plasmid is
cea
+
kil
-
imm
+
, so the damage can be attributed solely to the Kil function and not to the presence of colicin. However, cells carrying a
cea
+
kil
-
imm
-
plasmid are killed upon induction, apparently by action of endogenous colicin on the nonimmune cytoplasmic membrane. The pattern of accompanying physiological damage is distinguished from the
kil
+
-associated damage by an enhancement of α-methyl glucoside uptake and accumulation and efflux of α-methyl glucoside 6-phosphate and by an absence of the alteration in membrane permeability for
o
-nitrophenyl β-D-galactoside. These features are typical of colicin E1 action on the membrane. The induced damage is not prevented by trypsin and occurs in cells of a strain specifically tolerant to exogenous colicin E1, indicating that the attack is from inside the cell.
Replication and expression of Xenopus laevis globin genes injected into fertilized Xenopus eggs.
Authors: Bendig M M , Williams J G
Published: May 2006

Cloned Xenopus adult alpha 1- and beta 1-globin genes were injected into fertilized Xenopus eggs, and the eggs were allowed to develop into swimming tadpoles. The injected DNA replicated during early Xenopus development but did not become methylated de novo. When DNA was modified with Hpa II methylase before injection, methylation was maintained during replication. Although the Xenopus adult globin genes are not normally expressed until metamorphosis, both the unmethylated and in vitro methylated adult alpha- and beta-globin genes were transcribed at low levels, but from the correct promoters, during early development.
Uvomorulin: a nonintegral membrane protein of early mouse embryo.
Authors: Peyriéras N , Hyafil F , Louvard D , Ploegh H L , Jacob F
Published: May 2006

A monoclonal antibody has allowed the characterization of various forms of uvomorulin, a glycoprotein involved in the process of compaction of mouse morula. In addition to various degradation products, uvomorulin exists as a 120-kilodalton exocellular molecule stable at the cell surface. A short-lived 135-kilodalton precursor of uvomorulin has been detected after 10-min pulse labeling. Uvomorulin-like molecules are found on various tissues at various stages of development of the mouse.
Neurofilaments contain alpha-melanocyte-stimulating hormone (alpha-MSH)-like immunoreactivity.
Authors: Dräger U C , Edwards D L , Kleinschmidt J
Published: May 2006

An antiserum to alpha-melanocyte-stimulating hormone (alpha-MSH) was found to contain antibodies to at least two types of determinants on the alpha-MSH peptide: one is present only on the free peptide, the other is shared with neurofilaments. Immunoblots from mouse brain showed the neurofilament crossreactivity to be located on proteins in the Mr 140,000 range. The neurofilament-crossreactive portion of the antiserum could be selectively absorbed out with a cytoskeletal preparation, which abolished all affinity of the antiserum to the retina but did not affect the labeling pattern in the pituitary. Absorptions with desacetyl-alpha-MSH and corticotropin seemed to indicate that the determinant shared with neurofilaments is not located at either end of the alpha-MSH peptide, but somewhere in between. The immunohistochemical labeling of the retina with the alpha-MSH antiserum was compared to the labeling with monoclonal antibodies against Mr 200,000 neurofilaments. In the adult retina the alpha-MSH-like immunoreactivity was found to be slightly more widespread; most consistently it was detectable in cell bodies of large ganglion cells, whereas the heavy neurofilament subunit was absent from somata and proximal axons of these cells. In the developing mouse brain, expression of the heavy subunit was found to lag 2-3 wk behind expression of the Mr 140,000 proteins. This confirms previous reports of a more restricted distribution and late expression of high molecular weight neurofilaments as compared to the lower subunits.
tRNA transport from the nucleus in a eukaryotic cell: carrier-mediated translocation process.
Authors: Zasloff M
Published: May 2006

The mechanism by which a tRNA molecule is delivered from the nucleus of a cell to the cytoplasm has been studied in the Xenopus laevis oocyte utilizing nuclear microinjection and manual microdissection techniques. tRNA nuclear transport in this cell resembles a carrier-mediated translocation process rather than diffusion through a simple pore or channel. tRNA transport is saturable by tRNA, with a maximal rate measured to be about 190 X 10(7) molecules per min per nucleus (21 degrees C) in the mature oocyte. Competitive inhibition between two different tRNA species can be demonstrated, suggesting that many tRNA species share a common carrier system. tRNA nuclear transport is sharply dependent on temperature, with an optimal rate observed at 31 degrees C. A single G-to-U substitution at position 57 in the vertebrate tRNAMeti molecule reduces the transport rate of this tRNA by a factor of about 20, implicating this highly conserved region of the tRNA molecule (loop IV) as critical for recognition by the transport mechanism. On morphologic grounds I propose that ribosome-like components surrounding the nuclear pore may function as the tRNA translocation "motor." The tRNA nuclear transport mechanism represents a distinctly eukaryotic process and a site of potential control over cell growth and proliferation.
Seminal vesicle secretion IV gene: allelic difference due to a series of 20-base-pair direct tandem repeats within an intron.
Authors: Harris S E , Mansson P E , Tully D B , Burkhart B
Published: May 2006

The rat seminal vesicle secretion IV (SVS IV) gene was isolated from a lambda Charon 4A library. The SVS IV gene transcription unit was found to be on one 3.3-kilobase (kb) EcoRI fragment. Restriction mapping and DNA sequence analysis demonstrated that the entire length of the SVS IV transcription unit is 1,930 base pairs (bp) and contains two introns. The 3.3-kb EcoRI fragment contains 144 bp of 5'-flanking region. At -113 bp from the presumed transcription initiation site an interesting structure with perfect dyad symmetry is noted. In another lambda clone, a 3.5-kb EcoRI fragment was isolated that contains the SVS IV gene and was shown to be identical to the 3.3-kb EcoRI fragment except for 180 bp of DNA in the second intron. The extra DNA consists of several (8-10) 20-bp tandem repeats flanked on each side by seven or eight copies of this same 20-bp repeat. Fisher X Sprague-Dawley hybrid rats, which contain both the EcoRI 3.5-kb form and the 3.3-kb form of the SVS IV gene, were crossed with each other. Analysis of the F1 generation demonstrated that the presence or absence of the 180-bp intronic insertion in the SVS IV gene defines an allelic difference. This report also presents the DNA sequence of the transcription unit and flanking regions of the SVS IV gene.
Isolation of yeast DNA replication mutants in permeabilized cells.
Authors: Kuo C , Nuang H , Campbell J L
Published: May 2006

A random population of temperature-sensitive mutants was screened by assaying for defects in DNA synthesis in a permeabilized yeast DNA replication system. Twenty mutants defective in in vitro DNA synthesis have been isolated. In this paper we describe eight of these mutants. Seven of them fall into three complementation groups--cdc2, cdc8, and cdc16--involved in the control of the cell-division cycle. Because synthesis in vitro represents propagation of replication forks active in vivo at the time of permeabilization, our finding that cdc2 and cdc16 mutants can incorporate dTMP into DNA in such permeabilized cells at 23 degrees C but not at 37 degrees C supports the conclusion that these two mutations directly affect DNA synthesis at replication forks. Such an involvement was previously suggested by in vivo analysis for CDC2 but was less clear for CDC16. Finally, the usefulness of our screening procedure is demonstrated by the isolation of replication mutants in previously undescribed complementation groups. One strain shows a serious defect in in vivo DNA synthesis but normal RNA synthesis.
Complete DNA methylation does not prevent polyoma and simian virus 40 virus early gene expression.
Authors: Graessmann M , Graessmann A , Wagner H , Werner E , Simon D
Published: May 2006

The effect of DNA methylation on polyoma virus and simian virus 40 gene expression was investigated. For this purpose, the cytosines of all C-G dinucleotides of the viral DNAs were methylated by the use of rat liver methylase and the completeness of methylation was verified by dinucleotide analysis and restriction endonuclease treatment. The biological activity of unmethylated and fully methylated DNAs was tested by microinjecting them into tissue culture cells. The functions analyzed included early and late viral gene expression, viral DNA replication, oncogenic transformation efficiency, and virus maturation. No difference in any of these biological functions was observed between methylated and unmethylated DNA. Early gene expression of methylated DNA is not the result of demethylation because viral DNA reextracted from the injected cells, under nonpermissive conditions, retained the methylation pattern of the input DNA. In contrast, viral DNA extracted from transformed cells or from intact virus particles was partially or completely demethylated.
Keratin synthesis in normal mouse epithelia and in squamous cell carcinomas: evidence in tumors for masked mRNA species coding for high molecular weight keratin polypeptides.
Authors: Winter H , Schweizer J
Published: May 2006

Transplantable mouse squamous cell carcinomas (SCC), originally derived either from back skin or forestomach epithelium, do not synthesize high molecular weight keratin polypeptides [greater than 60 kilodaltons (kDa)] involved in the process of terminal differentiation in the corresponding normal tissues. The in vivo tumor keratin spectra consist of only low molecular weight keratin subunits at 60, 58, 52, 50, 47, and 46 kDa, each encoded by its own mRNA and encountered also in normal epidermis and forestomach epithelium. In addition, both tumors express a mRNA-dependent 40-kDa protein, whereas a 56-kDa protein and its mRNA are selectively found only in the forestomach tumor. Translation of mRNAs from both tumors in a cell-free system does not only generate analogues of the in vivo tumor keratin polypeptides, but also both SCC possess an additional mRNA coding in vitro for a 67-kDa keratin subunit that is not expressed, however, in the carcinomas in vivo. The identity of this in vitro synthesized keratin member with a 67-kDa keratin polypeptide of both normal epidermis and forestomach epithelium was confirmed by comparison of charge properties and peptide mapping. With regard to this particular keratin polypeptide, the tumors are obviously able to sequester the polypeptide's mRNA in an untranslatable state in the cells.
Structural homology of corticotropin-releasing factor, sauvagine, and urotensin I: circular dichroism and prediction studies.
Authors: Pallai P V , Mabilia M , Goodman M , Vale W , Rivier J
Published: May 2006

Three recently isolated peptides, whose sequences have been determined--the corticotropin (adrenocorticotropic hormone)-releasing factor of ovine origin, sauvagine, from the skin of the frog Phyllomedusa sauvagei, and urotensin I from the teleost fish, Catostomus commersoni--show high (greater than 50%) sequence homology. CD spectra of the three peptides in trifluoroethanol indicate predominantly helical character for these peptides. Analysis of the secondary structures by the Chou-Fasman method predicts that the overall structural organization of the peptides is the same. All three possess a long internal helix, spanning about 25 residues, connected by a turn region to a COOH-terminal structural element that is an alpha-helix in corticotropin-releasing factor and urotensin I and a beta-sheet in sauvagine. The values for helical content estimated from the prediction method agree reasonably well with those computed from the CD spectra. This agreement as well as the CD spectra of corticotropin-releasing factor fragment 5-33 support the specific assignments of helical regions derived from the Chou-Fasman analysis. The three peptides exhibit significantly less helical structure in water than in trifluoroethanol as indicated by CD spectra. Hydrophilicity profiles provided comparison of the three peptides in terms of their overall hydrophilicity and the location of the regions of maximal hydrophilicity. A unique distribution of hydrophilic and hydrophobic residues within the internal helices is revealed by helical wheel analysis. Patches of both types of residues are formed following a heptad (four/three) rule. Since the two patches are shifted by one residue relative to one another, together they occupy only one face of the helical surface, a feature distinct from other amphiphilic structures.
Plasma membrane retrieval in neoplastic pancreatic acinar cells.
Authors: Kanwar Y S , Rosenzweig L J , Jakubowski M L , Reddy J K
Published: May 2006

The dynamics of plasma membrane retrieval has been studied in the pancreatic acinar carcinoma in order to determine if neoplastic cells exhibiting a heterogeneity of cytodifferentiation states retain the capacity to interiorize and recycle plasma membrane. To this end, the plasma membranes of neoplastic pancreatic acinar cells were labeled with radioiodinated cationic ferritin (125I-CF), and the fate of the tracer was monitored by quantitative electron microscopic autoradiography. The undifferentiated granule-deficient cells of the tumor internalized membrane-bound 125I-CF and sequestered it predominantly in lysosomes. By contrast, the differentiated granule-rich cells internalized significantly more membrane-bound 125I-CF, and the tracer was localized in secretory granules and in lysosomes. The data suggest that neoplastic cells retain the capability of retrieving plasma membrane and that the dynamics of the process is correlated with the state of cytodifferentiation of the neoplastic cells.
Different 3' end points of deletions causing delta beta-thalassemia and hereditary persistence of fetal hemoglobin: implications for the control of gamma-globin gene expression in man.
Authors: Tuan D , Feingold E , Newman M , Weissman S M , Forget B G
Published: May 2006

DNA at the end point of the gene deletion associated with one form of hereditary persistence of fetal hemoglobin (HPFH) was cloned and used as a probe in gene mapping experiments to analyze the extent and approximate 3' end points of various deletions associated with HPFH and delta beta-thalassemia. The deletions in the two known forms of deletion-type HPFH were shown to be considerably more extensive than in the two cases of delta beta-thalassemia studied. The overall extents of the deletions in the two types of HPFH were quite similar in both cases and the 3' end points were located at a minimum distance of approximately equal to 52 and 57 kilobases from the 3' extremity of the beta-globin gene. In contrast, the 3' end points of the deletions in the two forms of delta beta-thalassemia were located approximately equal to 5 and 10 kilobases to the 3' side of the beta-globin gene. The extent of these deletions and the nature of the DNA brought into the vicinity of the gamma-globin genes by the deletions may therefore be a more important influence on the phenotype of the deletions than the specific nature of the DNA sequences that are deleted within the non-alpha-globin gene cluster as a result of the mutations.
Monoclonal antibodies against the antigen receptor on a cloned T-cell hybrid.
Authors: Samelson L E , Germain R N , Schwartz R H
Published: May 2006

A pigeon cytochrome c-specific and Ia molecule-restricted T-cell hybrid was used as an immunogen in order to obtain monoclonal anti-antigen receptor antibodies. Two antibodies were isolated that specifically bound to and inhibited interleukin (IL) 2 release from only the immunizing clone. Lectin-induced IL 2 release was not affected by these antibodies. Binding assays with purified and iodinated monoclonal antibody indicated that there were approximately equal to 25,000 binding sites on the T-cell hybrid. Immunoprecipitation and NaDodSO4/polyacrylamide gel electrophoresis of detergent lysates from surface-labeled hybrid cells revealed a heterodimeric structure composed of chains of apparent Mrs 45,000-50,000 and 40,000-44,000. The chains were linked by intermolecular disulfide bonds, and the difference in migration of the isolated chains under reducing and nonreducing conditions was consistent with the presence of intramolecular disulfide bonds. The molecule that has been identified is a candidate for the antigen-specific receptor on the immunizing T-cell clone.
Protease-sensitive regions in myosin subfragment 1.
Authors: Applegate D , Reisler E
Published: May 2006

Proteolytic digestions of myosin subfragment 1 (S-1) with elastase, subtilisin, papain, thermolysin, and Staphylococcus aureus protease reveal that the two trypsin-sensitive regions in S-1 have broad protease susceptibility. The cleavage of S-1 by these enzymes yields products that correspond within 1-2 kilodaltons (kDa) to the 25-, 50-, and 20-kDa fragments produced by trypsin. Papain and thermolysin cut preferentially at the 26-kDa/70-kDa junction, whereas elastase, subtilisin, and S. aureus protease cleave both the 26-kDa/70-kDa and 75-kDa/22-kDa junctions in S-1. Binding of actin to S-1 decreases the rate of all proteolytic reactions in the 95-kDa heavy chain. The protection of the 26-kDa/70-kDa junction by actin is greatest against papain and thermolysin attack. The reaction times of elastase, subtilisin, and S. aureus protease with S-1 increase 2-fold in the presence of actin. However, in contrast to similar reactions with trypsin, they proceed at both junctions and lead to formation of the 50- and 22-kDa fragments. The cleavage of the 22-kDa/50-kDa junction by elastase increases the Km value for the actin-activated ATPase. The presence of the two protease-sensitive regions in S-1 is consistent with a three-domain structure of the myosin head and may have important implications to the mode of intersite communication in this protein.
Arachidonate metabolism by human polymorphonuclear leukocytes stimulated by N-formyl-Met-Leu-Phe or complement component C5a is independent of phospholipase activation.
Authors: Clancy R M , Dahinden C A , Hugli T E
Published: May 2006

Release of arachidonic acid by the membrane phospholipase and metabolism by the 5-lipoxygenase pathway was examined in human polymorphonuclear leukocytes (PMNs). The 5-lipoxygenase pathway is activated when PMNs are given arachidonic acid in ethanol and there is extensive metabolism to 5-hydroxyicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4). This activation event was shown to be altered by the ethanol because resting PMNs given arachidonic acid with bovine serum albumin fail to metabolize arachidonic acid. However, cells activated by the inflammatory agents N-formyl-Met-Leu-Phe (fMLF) or complement component C5a recruit the 5-lipoxygenase to metabolize exogenous arachidonic acid to 5-HETE and LTB4. When PMNs were incubated with arachidonic acid-bovine serum albumin and challenged with fMLF or C5a (des-Arg-C5a) they produced 49-75 pmol of LTB4 and 310-440 pmol of 5-HETE per 10(7) cells. PMNs stimulated by fMLF or C5a (des-Arg-C5a) do not induce membrane phospholipases to mobilize endogenous arachidonic acid and neither 5-HETE nor LTB4 is formed. In contrast, PMN stimulation by the ionophore A23187 activates both the membrane phospholipase and the 5-lipoxygenase to produce 5-HETE and LTB4 from endogenous arachidonic acid. Our results indicate that the lipoxygenase pathway is inoperative in resting PMNs but can be recruited by chemotactic factors to act on arachidonate from extracellular sources. It was previously believed that formation of 5-HETE and LTB4 by the PMN depends solely on phospholipase to mobilize endogenous arachidonic acid. The results reported here refute this concept and indicate that the role of phospholipase activation in PMN may be overestimated. Therefore, subsequent involvement of lipoxygenase products in mediating stimulation of PMN by inflammatory factors (e.g., as in aggregation and chemotaxis) remains in question unless an exogenous source of arachidonate can be identified.
Cell cycle-dependent intervention by benzamide of carcinogen-induced neoplastic transformation and in vitro poly(ADP-ribosyl)ation of nuclear proteins in human fibroblasts.
Authors: Kun E , Kirsten E , Milo G E , Kurian P , Kumari H L
Published: May 2006

Human fibroblasts were subjected to nutritionally induced G1 block, followed by release and subsequent entry into S phase, and exposed to nontoxic concentrations of carcinogens in early S phase. Cell transformation occurred as determined by early morphologic cell alterations, anchorage-independent colony formation, cell invasiveness, and augmentation of Ab 376 human malignancy-specific cell-surface antigenic determinant. Methylazoxymethanol acetate was the most potent transforming agent at doses that were negative in toxicity tests. Benzamide (10 microM intracellular concentration), a specific inhibitor of poly(ADP-ribose) polymerase, prevented transformation in a cell cycle-specific manner, maximal prevention coinciding with early S phase, also characteristic of maximal susceptibility to transformation. Neither an interference of carcinogen deoxyguanosine nucleoside adduct formation nor a chemical reaction between benzamide and carcinogens was detected. Methylazoxymethanol acetate at transforming but nontoxic dose partially inhibited poly(ADP-ribosyl)ation to about the same extent as benzamide. However, simultaneous exposure of cells to both agents in early S phase, resulting in the prevention of transformation, augmented poly(ADP-ribosyl)ation above the controls. Enzymatic activities ran parallel with the formation of DNA-associating polymer-nonhistone protein adducts that are assumed to regulate the physiological function of chromatin at the structural level.
A synaptic vesicle antigen is restricted to the junctional region of the presynaptic plasma membrane.
Authors: Buckley K M , Schweitzer E S , Miljanich G P , Clift-O'Grady L , Kushner P D , Reichardt L F , Kelly R B
Published: May 2006

The plasma membrane of electric organ nerve terminals has two domains that can be distinguished by monoclonal antibodies. A library of 111 mouse monoclonal antibodies raised to nerve terminals from Torpedo californica contains 4 antibodies that bind specifically to the outside of intact synaptosomes. The distribution of the binding sites of these monoclonal antibodies on the outside of intact nerve terminals was examined by immunofluorescence and immunoelectron microscopy. The binding sites of 3 (tor23, 25, and 132) are distributed uniformly over nerve trunks and fine terminal branches. The binding site of the fourth (tor70) is restricted to synaptic junctional regions. This antibody, but not the other 3, recognizes a major component of synaptic vesicles, a proteoglycan associated with the inner surface of the vesicle membrane. The difference in the pattern of binding of these monoclonal antibodies suggests that the region of the plasma membrane containing active zones is antigenically distinguishable from other nerve terminal plasma membrane. We suggest that the antigen recognized by tor70 is externalized by exocytosis of synaptic vesicles while other plasma antigens take a different route to the surface. The unexpected observation that the vesicle antigen remains on the surface after exocytosis and is prevented from diffusion from the synaptic junctional region would be consistent with an interaction between the vesicle proteoglycan and elements of the synaptic cleft.
cDNA cloning of the bovine low density lipoprotein receptor: feedback regulation of a receptor mRNA.
Authors: Russell D W , Yamamoto T , Schneider W J , Slaughter C J , Brown M S , Goldstein J L
Published: May 2006

The low density lipoprotein (LDL) receptor belongs to a class of migrant cell surface proteins that mediate endocytosis of macromolecular ligands. No cDNAs for this class of proteins have been isolated to date. In the current paper, we report the isolation of a cDNA clone for the LDL receptor from a bovine adrenal cDNA library. The library was constructed by the Okayama-Berg method from poly(A)+ RNA that had been enriched in receptor mRNA by immunopurification of polysomes. Mixtures of synthetic oligonucleotides encoding the amino acid sequence of two neighboring regions of a single cyanogen bromide fragment were used as hybridization probes to identify a recombinant plasmid containing the LDL receptor cDNA. This plasmid, designated pLDLR-1, contains a 2.8-kilobase (kb) insert that includes a sequence which corresponds to the known amino acid sequence of a 36-residue cyanogen bromide fragment of the receptor. pLDLR-1 hybridized to a mRNA of approximately equal to 5.5 kb in the bovine adrenal gland. This mRNA, like the receptor protein, was 9-fold more abundant in bovine adrenal than in bovine liver. pLDLR-1 cross-hybridized to a mRNA of approximately equal to 5.5 kb in cultured human epidermoid carcinoma A-431 cells. This mRNA was markedly reduced in amount when sterols were added to the culture medium, an observation that explains the previously observed feedback regulation of LDL receptor protein. Southern blot analysis of bovine genomic DNA with 32P-labeled pLDLR-1 revealed a simple pattern of hybridization, consistent with a single-copy gene containing introns.
Developmental regulation of the Aspergillus nidulans trpC gene.
Authors: Yelton M M , Hamer J E , de Souza E R , Mullaney E J , Timberlake W E
Published: May 2006

We have cloned the trifunctional trpC gene from Aspergillus nidulans by hybrid phage lambda complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase activity. Four different phages sharing a 4.3-kilobase region were obtained. Plasmid subclones containing this region also complemented the E. coli trpC mutant. We determined that a 1.8-kilobase DNA fragment was minimally required for complementation. The fragment hybridized with two poly(A)+ RNAs, 3.0 and 3.2 kilobases in length. We infer that these transcripts are Aspergillus trpC mRNAs and that the entire Aspergillus trpC gene is not required for complementation in E. coli. Levels of both trpC transcripts in poly(A)+ RNA are regulated by growth medium composition. They were highest when cells were grown in minimal medium containing nitrate as the nitrogen source and lowest when cells were grown in medium containing yeast extract. The concentrations of the transcripts are also regulated during conidiophore development. Conidiating cultures grown on medium containing yeast extract had significantly higher levels of both transcripts than did hyphae grown in minimal medium containing nitrate. Levels of the transcripts in mature spores were equivalent to those found in hyphae grown in minimal medium containing nitrate. Results from nutritional experiments with an A. nidulans trpC mutant suggest that developmental regulation of trpC mRNA levels may be related to a high requirement for tryptophan or a compound derived from tryptophan during conidiation.
Purification and characterization of trp aporepressor.
Authors: Joachimiak A , Kelley R L , Gunsalus R P , Yanofsky C , Sigler P B
Published: May 2006

We have isolated homogeneous trp aporepressor from an overproducing strain of Escherichia coli carrying a plasmid containing trpR preceded by tandem trp operon promoters. Dye-affinity and ion-exchange chromatography were used in conjunction with a gel electrophoresis assay in which the repressor, when bound to the trp operator, protects an Rsa I restriction site from endonuclease cleavage. Crystals suitable for x-ray diffraction studies were grown from a variety of concentrated salt solutions. Hydrodynamic properties and electrophoretic analysis of unmodified and covalently crosslinked aporepressor show that the free aporepressor has an isoelectric point of 5.9 and is a dimer containing two identical 12.5-kilodalton subunits in the presence or absence of L-tryptophan. The repressor . operator complex binds poorly to nitrocellulose filters, but restriction-site protection studies indicate that, in the presence of tryptophan, one dimer is bound to the operator site with an apparent dissociation constant less than 2 X 10(-9) M. Preliminary equilibrium dialysis experiments suggest that tryptophan binds to the aporepressor with a dissociation constant of 1.6 X 10(-5) M.
Sterol synergism in yeast.
Authors: Ramgopal M , Bloch K
Published: May 2006

Sterol synergism as previously observed [Dahl, C.E., Dahl, J.S. & Bloch, K. (1980) Biochemistry 19, 1462-1467] and defined as a greater-than-additive growth response to pairs of sterols by Mycoplasma capricolum [Dahl, J.S., Dahl, C.E. & Bloch, K. (1981) J. Biol. Chem. 256, 87-91] is now demonstrated in the yeast mutant GL7, which is auxotrophic for sterol and unsaturated fatty acid. Mutant cells growing poorly when provided with cholesterol and oleic acid respond to ergosterol supplements (ergosterol-to-cholesterol ratio, 1:3) by a pronounced increase in growth rates and cell yields. Stigmasterol also elicits a significant synergistic effect, and 7-dehydrocholesterol, a smaller one. Evidence for a metabolic role of ergosterol in yeast membranes is presented. Cells raised on a 1:3 mixture of ergosterol to cholesterol up to midlogarithmic phase subsequently incorporate [1-14C]oleic acid at significantly faster rates into phospholipids than do cells grown on cholesterol alone.
X-ray scattering from the superhelix in circular DNA.
Authors: Brady G W , Fein D B , Lambertson H , Grassian V , Foos D , Benham C J
Published: May 2006

This communication presents measurements, made with a newly constructed position-sensitive detector, of the small-angle x-ray scattering from the first-order superhelix of native COP608 plasmid DNA. This instrument measures intensities free of slit effects and provides good resolution in the region of interest. The reported observations, made both in the presence and in the absence of intercalator, closely fit the scattering patterns calculated for noninterwound helical first-order superhelices. These results are consistent with a toroidal helical structure but not with interwound conformations. The pitch angle alpha and contour length per turn c are reported for the native molecule at several concentrations of the platinum intercalating compound. From these parameters, the best-fitting toroidal helix is constructed and its geometry is investigated. The specific linking difference of the native molecule is estimated to be delta Lk/Lk0 approximately equal to -0.055. If the best-fitting toroidal helix is taken to be the actual structure, the partitioning of superhelicity between twist and writhe occurs in the approximate ratio of 2:1.
Transcriptional activation of the translocated c-
myc
oncogene in Burkitt lymphoma
Authors: Erikson Jan , Ar-Rushdi Abbas , Drwinga Helen L. , Nowell Peter C. , Croce Carlo M.
Published: May 2006

We have previously demonstrated that translocations of

V
H

genes from chromosome 14 to chromosome 8 and of the c-
myc
oncogene from chromosome 8 to chromosome 14 occur in Burkitt lymphomas with the t(8;14) chromosome translocation. An association of the c-
myc
gene with the
C
μ
immunoglobulin gene has been observed in some but not all Burkitt lymphomas studied previously. In the present study, we have investigated the organization of the human heavy chain locus and of the c-
myc
gene in the P3HR-1 Burkitt lymphoma cell line. Becuase mouse/P3HR-1 somatic cell hybrids that retain only the 14q+ chromosome and no other human chromosome contain the human
C
μ
and
C
γ
genes but not

V
H

genes, we have concluded that the breakpoint on chromosome 14 in P3HR-1 cells is distal to
C
μ
and between
C
μ
and

V
H

. Thus, the breakpoint of human chromosome 14 differs in different Burkitt lymphoma cell lines. We also found that the human c-
myc
oncogene translocated to chromosome 14 in the P3HR-1 cell line is not recombined with the
C
μ
gene. The breakpoint on human chromosome 8 may therefore also differ in different Burkitt lymphoma cell lines, because we have observed DNA rearrangement of the c-
myc
gene with the
C
μ
gene in only some of the Burkitt lymphoma cell lines studied elsewhere. Interestingly, high levels of transcripts of the c-
myc
oncogene were observed in Burkitt lymphomas with translocated c-
myc
oncogenes both rearranged and unrearranged. Therefore, the translocation of a c-
myc
oncogene to the heavy chain locus on human chromosome 14 is apparently sufficient for its transcriptional activation and may be an essential step in the pathway leading to neoplasia.
Anti-idiotypic antibodies to anti-HLA receptors induced by pregnancy
Authors: Suciu-Foca N. , Reed E. , Rohowsky C. , Kung P. , King D. W.
Published: May 2006

We have previously shown that lymphoblasts alloactivated
in vitro
acquire the capacity of stimulating the autologous mixed lymphocyte response. This response is anti-idiotypic in nature because lymphocytes so primed display accelerated memory responses only when restimulated by autologous lymphoblasts that have been alloactivated against the same HLA-DR antigen. Based on this observation we have postulated that the absence of HLA antibodies in alloimmunized human subjects may be due to the development of autoantibodies that react with the anti-HLA receptors expressed by primed lymphocytes or by anti-HLA antibodies or both. This hypothesis has been confirmed in the present investigations which show that sera from parous women react with autologous T lymphoblasts primed in 5-day mixed lymphocyte culture against their husband—i.e., with lymphoblasts expressing receptors for the immunizing donor. Anti-HLA receptors expressed by T and B lymphocytes seem to share serologic determinants because sera that bind to autologous alloactivated lymphoblasts are also capable of inhibiting the anti-HLA activity of autologous and homologous sera. Auto-anti-idiotypic antibodies inhibit the autologous mixed lymphocyte response to autologous alloactivated lymphoblasts, a phenomenon whose
in vivo
correlate may reside in autoinhibition of anti-HLA antibody formation and of allograft immunity. Because auto-anti-idiotypic antibodies were found in sera from all parous women tested, the hypothesis that nonresponsiveness to alloantigens exists as a state
per se
is not likely. The passive transfer of antireceptor (idiotype) immunity by use of antibodies from pregnant women's sera may provide a powerful tool for specific suppression of allograft rejection.