bluuebunny/crossref_metadata_2025 huggingface dataset:
Adaptive resynthesis of
O
6
-methylguanine-accepting protein can explain the differences between mammalian cells proficient and deficient in methyl excision repair
Authors: Waldstein E. A. , Cao E.-H. , Setlow R. B.
Published: May 2006

Mammalian cells have been classified as proficient (Mer
+
) or deficient (Mer
-
) in methyl excision repair in terms of their cytotoxic reactions to agents that form
O
6
-alkylguanine and their abilities to reactivate alkylated adenoviruses.
O
6
-Methylguanine (
O
6
MeGua) is considered to be a lethal, mutagenic, and carcinogenic lesion. We measured the abilities of cell extracts to transfer the methyl group from an exogenous DNA containing
O
6
MeGua to acceptor protein. The constitutive level of acceptor activity was independent of the Mer phenotype and was ≈ 100,000 acceptor sites per cell. Treatment of cells with
N
-methyl-
N
′-nitro-
N
-nitrosoguanidine (MNNG) results in a dose-dependent decrease in the acceptor activity in extracts because the rapid reaction between endogenous
O
6
MeGua and acceptor protein makes the latter unavailable for further reaction. Treatment of cells with 1 μM MNNG for 15 min or 2 μM for ≈2 min uses up >95% of the constitutive activity. However, Mer
+
cells, which are resistant to MNNG, rapidly resynthesize new acceptor protein, and the activity returns to the basal level in ≈90 min. In Mer
-
tumor cells and Chinese hamster cells, which are sensitive to MNNG, resynthesis is not detectable in 90 min. Mer
-
simian virus 40-transformed fibroblasts, known to have an intermediate sensitivity to MNNG, have an intermediate resynthesis rate. Treatment of cells with multiple low doses of MNNG results in the enhanced production of
O
6
MeGua-accepting protein in levels 2.5-fold above the constitutive values for Mer
+
tumor cells and to ≈ 1.5-fold for Mer
+
fibroblasts or Mer
-
simian virus 40-transformed cells. Such treatments reduce the activities in Mer
-
tumor cells and Chinese hamster cells. We conclude: (
i
) estimates of
O
6
MeGua in cellular DNA shortly after treatment may be seriously in error because of the rapid repair of this lesion, and (
ii
) the adaptive resynthesis of acceptor protein, not its constitutive level, is the important correlate of cell resistance to methylating agents.
Sequence of a cDNA encoding pancreatic preprosomatostatin-22.
Authors: Magazin M , Minth C D , Funckes C L , Deschenes R , Tavianini M A , Dixon J E
Published: May 2006

We report the nucleotide sequence of a precursor to somatostatin that upon proteolytic processing may give rise to a hormone of 22 amino acids. The nucleotide sequence of a cDNA from the channel catfish (Ictalurus punctatus) encodes a precursor to somatostatin that is 105 amino acids (Mr, 11,500). The cDNA coding for somatostatin-22 consists of 36 nucleotides in the 5' untranslated region, 315 nucleotides that code for the precursor to somatostatin-22, 269 nucleotides at the 3' untranslated region, and a variable length of poly(A). The putative preprohormone contains a sequence of hydrophobic amino acids at the amino terminus that has the properties of a "signal" peptide. A connecting sequence of approximately 57 amino acids is followed by a single Arg-Arg sequence, which immediately precedes the hormone. Somatostatin-22 is homologous to somatostatin-14 in 7 of the 14 amino acids, including the Phe-Trp-Lys sequence. Hybridization selection of mRNA, followed by its translation in a wheat germ cell-free system, resulted in the synthesis of a single polypeptide having a molecular weight of approximately 10,000 as estimated on Na-DodSO4/polyacrylamide gels.
Carcinogen-transformed human cells are inhibited from entry into S phase by fusion to senescent cells but cells transformed by DNA tumor viruses overcome the inhibition.
Authors: Stein G H , Yanishevsky R M , Gordon L , Beeson M
Published: May 2006

Senescent human diploid cells (HDC) were fused to replicative transformed cells of different types, and DNA synthesis was monitored in the resulting heterodikaryons. Human cells transformed by simian virus 40 or adenovirus serotype 5 were able to induce DNA synthesis in senescent HDC nuclei in heterodikaryons. In contrast, carcinogen-transformed cells were not able to induce DNA synthesis in senescent HDC nuclei; rather, the transformed nuclei in these heterodikaryons were inhibited from entering S phase. Cells transformed by Rous sarcoma virus and most human tumor cells tested are similarly inhibited by fusion to senescent HDC. These results suggest that the mechanism for transformation by DNA tumor viruses may be fundamentally different from that of other viruses and carcinogens and from that of most human tumor cells. A simple model to explain these results is that (i) senescent HDC contain an inhibitor of entry into S phase; (ii) cells transformed by DNA tumor viruses have gained a transforming factor, perhaps large tumor antigen, that is capable of overriding the normal inhibitor; and (iii) cells transformed by carcinogens or RNA viruses have lost or altered the mechanism for expression of the normal inhibitor yet are still sensitive to it. We propose that this inhibitor is produced in normal cells when they experience conditions that are inadequate for proliferation and that it plays a role in putting the cells into a distinct quiescent state with long-term viability. The override of this inhibitor function in simian virus 40-transformed HDC can explain why they have low viability in plateau-phase cultures and why they die during crisis.
Immunofluorescent localization of the transforming protein of Rous sarcoma virus with antibodies against a synthetic src peptide.
Authors: Nigg E A , Sefton B M , Hunter T , Walter G , Singer S J
Published: May 2006

Antisera were raised against a synthetic peptide (src-c) containing the six COOH-terminal amino acids of p60src, the transforming protein of Rous sarcoma virus (RSV). Antibodies specific for the src-c peptide were purified by affinity chromatography and then used to study the location of p60src in transformed cells. The distribution of p60src was compared to that of vinculin, a candidate cytoskeletal substrate of p60src, by indirect double immunofluorescence microscopy. In RSV-transformed rat, mouse, and chicken cells, an extensive codistribution of p60src with vinculin was observed. Both proteins were concentrated in the few remaining focal adhesion plaques, in transformation-induced rosette clusters at the ventral cell surface, and in cell-cell contact areas. In addition, antibodies to both proteins stained the cytoplasm diffusely. In all cells examined, the immunofluorescent staining patterns produced by antibodies to the src-c peptide were indistinguishable from those obtained by immunolabeling of p60src with sera from RSV-infected tumor-bearing rabbits. The excellent agreement of the results obtained with two completely independent antibody preparations indicates strongly that the observed immunolabeling patterns correctly define the intracellular distribution of p60src. The significance of the intracellular location of p60src to the transforming activities of the protein is discussed.
Periportal and pericentral pyridine nucleotide fluorescence from the surface of the perfused liver: evaluation of the hypothesis that chronic treatment with ethanol produces pericentral hypoxia.
Authors: Ji S , Lemasters J J , Christenson V , Thurman R G
Published: May 2006

Pyridine nucleotide fluorescence made from the surface of the hemoglobin-free perfused rat liver was measured continuously by using a "micro-light guide" placed on selected periportal and pericentral regions of the liver lobule. From the portal oxygen tension at which pyridine nucleotide reduction first occurred in pericentral regions, the oxygen gradient across the liver lobule was estimated in livers from rats treated chronically with ethanol or sucrose. Chronic treatment with ethanol increased the average lobular oxygen gradient from 275 to 400 torr (1 torr = 133 Pa), primarily due to the increase in the oxygen gradient in pericentral regions. Ethanol treatment also increased hepatic oxygen uptake significantly, from 110 to 144 (mumol/g)/hr. Treatment with the antithyroid drug 6-propyl-2-thiouracil reversed the effect of ethanol on O2 uptake and on the lobular oxygen gradient. The oxygen gradients measured with the micro-light guide were confirmed by direct measurement of tissue oxygen tensions in periportal and pericentral areas by using an oxygen electrode. These data are consistent with the hypothesis that chronic treatment with ethanol causes the pericentral region of the liver lobule to become susceptible to hypoxic cellular injury. This may be responsible, at least in part, for the localized hepatotoxic effects of ethanol.
Isolation and amino acid sequence analysis of a 4,000-dalton dynorphin from porcine pituitary.
Authors: Fischli W , Goldstein A , Hunkapiller M W , Hood L E
Published: May 2006

A 4,000-dalton dynorphin was isolated from porcine pituitary. It has 32 amino acids (Mr = 3,986), with the previously described heptadecapeptide (now called dynorphin A) at its amino terminus and a related tridecapeptide, dynorphin B, at its carboxyl terminus. The two peptides are separated by the "processing signal" Lys-Arg.
Redistribution of amylase activity accompanying its secretion by the pancreas.
Authors: Miyasaka K , Rothman S S
Published: May 2006

Amylase activity in several tissue and body fluid compartments in the rat changed markedly when the secretion of digestive enzyme was augmented over a 3-hr period with a cholinergic agonist. As a result of stimulation, the pancreas was depleted of about one-third of its amylase activity and accounted for only 75% of the amount recovered from the animal, compared to 92% in the fasted state. Despite the continuous augmented secretion of the enzyme into the small intestine, no increase in amylase activity was detected there at the end of 3 hr. On the other hand, amylase activity in plasma and extracellular fluid increased by about an order of magnitude and accounted for 13% of the total pool, compared to approximately 1% in the fasted state. Amylase activity in several solid tissues also increased, including a 50- to 100-fold increase in parotid gland and an almost 10-fold increase in submandibular gland and kidney. The potential sources of the increased amylase activity in blood, the endocrine secretion of the enzyme by the pancreas, and its absorption from the intestine are considered. Changes in the amylase content of various tissues appear to reflect increased uptake due to increased plasma levels.
Delayed clearance of very low density and intermediate density lipoproteins with enhanced conversion to low density lipoprotein in WHHL rabbits.
Authors: Kita T , Brown M S , Bilheimer D W , Goldstein J L
Published: May 2006

Rabbit livers express two genetically distinct receptors for plasma lipoproteins: (i) the low density lipoprotein (LDL) receptor and (ii) the chylomicron remnant receptor. In homozygous Watanabe-heritable hyperlipidemic (WHHL) rabbits, an animal model for human familial hypercholesterolemia, LDL receptors are genetically deficient, but chylomicron remnant receptors are normal. Hence, WHHL rabbits clear LDL from the circulation at an abnormally slow rate, but they clear chylomicron remnants at a normal rate. The current studies show that WHHL rabbits clear 125I-labeled very low density lipoprotein (VLDL) and its metabolic product, intermediate density lipoprotein (IDL), from plasma at a markedly decreased rate. The impaired clearance is due to a profound decrease in the rate of uptake of 125I-labeled VLDL and 125I-labeled IDL by the liver. Because of its rapid clearance in normal rabbits, only a fraction of the 125I-labeled apoprotein B component of VLDL is converted to LDL. In WHHL rabbits, the impaired clearance of VLDL leads to a markedly increased conversion of 125I-labeled apoprotein B from VLDL to LDL. These results indicate that: (i) in rabbits, the LDL receptor mediates the rapid removal of VLDL and IDL from plasma, and (ii), a deficiency of LDL receptors leads to an enhanced conversion of VLDL to LDL. The combination of overproduction and impaired plasma clearance of LDL, both resulting from a single gene mutation in the LDL receptor, leads to a massive increase of plasma LDL levels in homozygous WHHL rabbits.
Proximity of the substrate binding site and the heme-iron catalytic site in cytochrome P-450scc.
Authors: Sheets J J , Vickery L E
Published: May 2006

As an approach to "mapping" the active site of the cytochrome P-450 that catalyzes cholesterol side-chain cleavage, designated cytochrome P-450scc, we have synthesized steroid derivatives with the potential to interact with both the substrate binding site and the heme-iron catalytic site of the enzyme. The effects of these substrate analogs were studied with cytochrome P-450scc purified from bovine adrenal cortex. One derivative, 22-amino-23,24-bisnor-5-cholen-3 beta-ol, was found to be a potent inhibitor of pregnenolone formation in a reconstituted enzyme system, and a kinetic analysis of the inhibition showed that binding of the derivative is competitive with respect to cholesterol. The spectral properties of a stable complex formed between the steroidal amine and the purified cytochrome suggest that the 22-amine group coordinates directly to the heme-iron. A model for the structure of this inhibitor-enzyme complex is proposed in which the 5-androstene ring system of the steroid occupies the substrate binding site, and the amine group of the side chain occupies an axial coordination position of the Fe(III) center. This places limits on the distance between these two domains in the enzyme and offers support for proposed mechanisms of cytochrome P-450-catalyzed oxygen-insertion reactions in which an iron-bound oxidant directly attacks the substrate.
Messenger RNA species partially in a repressed state in mouse sarcoma ascites cells.
Authors: Yenofsky R , Bergmann I , Brawerman G
Published: May 2006

Four major mRNA species of mouse sarcoma ascites cells, coding for polypeptides designated P65, P40, P36, and P21, occur predominantly as untranslated messenger ribonucleoprotein particles. Cloned cDNA probes were used to study their distribution in cytoplasmic extracts of these cells. A considerable portion of the mRNA molecules sedimented as small particles, whereas the rest was present in polyribosomes. In contrast, the actin mRNA was present almost exclusively in polyribosomes. Incubation of the ascites cells in culture medium, particularly after a starvation treatment, caused an enhancement in polypeptide chain initiation relative to elongation in these cells, as evidenced by a shift of ribosomes into the polyribosome fraction and by an increase in polyribosome size. Exposure of the cells to a low concentration of cycloheximide, an inhibitor of the elongation step, had a similar effect. The actin mRNA and the active P65, P40, P36, and P21 mRNA molecules were shifted to larger polyribosomes in the treated cells, but no shift of molecules from small particles to polyribosomes was observed. The incubation in culture also led to considerable increases in the proportion of P65 and P40 mRNA molecules in the untranslated state. The results indicate that the untranslated state cannot be attributed to poor initiation efficiency. It is suggested that a portion of the mRNA molecules is maintained in a repressed state and that mRNA repression may represent an important translation control process.
Instability of coated vesicles in concentrated sucrose solutions.
Authors: Nandi P K , Irace G , Van Jaarsveld P P , Lippoldt R E , Edelhoch H
Published: May 2006

A method of preparing homogeneous coated vesicles that eliminates the high sucrose concentrations heretofore used is presented. It is shown that sucrose at high concentrations dissociates the coat from coated vesicles. This reaction can explain the presence of empty coats observed with preparations obtained with high concentrations of sucrose. The protein and membrane lipid components have been analyzed by the intrinsic tryptophan and extrinsic diphenylhexatriene fluorescence, respectively. Analysis of mixtures of coated vesicles and baskets resolved the contributions of the two species to the fluorescence curves.
Encapsidation sequences for spleen necrosis virus, an avian retrovirus, are between the 5' long terminal repeat and the start of the gag gene.
Authors: Watanabe S , Temin H M
Published: May 2006

The minimal cis-acting sequences outside the long terminal repeat (LTR) required for formation of an infectious retrovirus cloning vector were determined with recombinants of spleen necrosis virus (SNV) DNA and herpes simplex virus type 1 thymidine kinase gene. The 3' end of SNV DNA was removed to within 40 base pairs (bp) from the 3' LTR with only a 2-fold effect on the recovery of infectious recombinant virus. However, when the 5' end of SNV DNA was removed to within 100 bp from the 5' LTR, infectious recombinant virus was not recovered. Deletion mutants constructed around this latter region showed that nucleotides between 100 and 285 bp from the 5' LTR are necessary for encapsidation of genomic viral RNA. We call this region required for encapsidation E.
The amino acid sequence and gene organization of the heavy chain of the HLA-DR antigen: homology to immunoglobulins.
Authors: Korman A J , Auffray C , Schamboeck A , Strominger J L
Published: May 2006

The amino acid sequence of the heavy chain of HLA-DR antigens has been elucidated from the analysis of a genomic clone coding for this protein. A 3.2-kilobase EcoRI fragment includes four exons containing 227 amino acids out of 229 in the mature HLA-DR heavy chain. One exon (alpha 2) encodes a domain of 94 amino acids with strong sequence homology both to Ig constant region domains and to Ig-like domains in HLA-B7, beta 2-microglobulin, and the HLA-DR light chain. These results support a structure for the HLA-DR antigen heterodimer consisting of four extracellular domains, two of which are Ig-like [one in the heavy chain (alpha 2) and one in the light chain (beta 2)]. The third is the amino-terminal polymorphic domain in the light chain (beta 1), and the fourth is an invariant domain in the heavy chain (alpha 1).
Reconstitution after transplantation with T-lymphocyte-depleted HLA haplotype-mismatched bone marrow for severe combined immunodeficiency.
Authors: Reinherz E L , Geha R , Rappeport J M , Wilson M , Penta A C , Hussey R E , Fitzgerald K A , Daley J F , Levine H , Rosen F S , Schlossman S F
Published: May 2006

Severe combined immunodeficiency (SCID) is potentially correctable by bone marrow transplantation if a patient has a suitable histocompatible donor. In the absence of an HLA-matched donor, lethal graft-versus-host disease (GVHD), which is mediated by alloreactive donor T cells, may occur. In an attempt to prevent GVHD in one SCID patient lacking a matched donor, we treated maternal haplomismatched bone marrow with a unique nonmitogenic T-cell-specific monoclonal antibody (anti-T12) and complement to remove mature T cells. Despite the removal of greater than 99% mature T cells, the child developed significant life-threatening GVHD, which was terminated by a 5-day course of intravenous anti-T12. Subsequently, immune reconstitution occurred by 6 wk: the mature circulating T cells proliferated in response to soluble and allo-antigens in vitro and provided help for B-cell immunoglobulin synthesis. The patient was removed from a protective environment and discharged without evidence of further infection. Both HLA and chromosomal analyses showed that the circulating cells in the patient were of maternal origin. More importantly, the maternal T cells were no longer reactive with recipient cells. Mixing experiments indicated that the state of tolerance that resulted in this chimera was not due to active suppression. We conclude that HLA-mismatched transplantation for SCID can be undertaken if mature alloreactive donor T lymphocytes are depleted before and after bone marrow grafting.
Sleep-promoting effects of muramyl peptides.
Authors: Krueger J M , Pappenheimer J R , Karnovsky M L
Published: May 2006

A muramyl peptide that induces excess slow-wave sleep in rats, rabbits, and cats has recently been isolated from human urine. We now report that synthetic acetylmuramyl-L-alanyl-D-isoglutamine ("muramyl dipeptide") and its lysyl derivative (acetylmuramyl-L-alanyl-D-isoglutaminyllysine) can mimic the somnogenic effects of the natural peptide. Both compounds are also pyrogenic and may cause other disturbances of autonomic function. The pyrogenic effects of intravenously administered muramyl dipeptide can be suppressed by previous treatment with acetaminophen without blocking the sleep-promoting effects.
Sexual dimorphism in Ramapithecinae
Authors: Kay Richard F.
Published: May 2006

The Ramapithecinae are an extinct, mainly Miocene group of hominoids comprising the genera
Sivapithecus
and
Gigantopithecus. Ouranopithecus
and
Ramapithecus
are other included genera, here regarded as invalid. Cladistically, ramapithecines are hominid, although, in most aspects of their anatomy, they remain very primitive or ape-like. Miocene ramapithecines show reduced sexual dimorphism in canine size. In this respect they resemble Pliocene/Recent hominids, not extant great apes (which have highly dimorphic canines). Reduced dimorphism in canine size is an important shared derived feature indicating the hominid status of ramapithecines. Among living anthropoids, a significant association has been observed between a monogamous social structure and low canine dimorphism. This supports the inference that ramapithecines may have been monogamous.
Posttranscriptional control of bacteriophage lambda gene expression from a site distal to the gene.
Authors: Guarneros G , Montañez C , Hernandez T , Court D
Published: May 2006

The bacteriophage lambda int gene product, integrase, recombines the phage DNA with the host DNA at specific sites on each to accomplish lysogeny. The int gene is transcribed from two promoters, PL and PI, each regulated positively by lambda proteins. The expression of integrase is also controlled from a site, sib, in the b region of the phage genome. This is a unique regulatory site because it is located distal to the structural gene in relation to the promoters. The expression of int from the PL promoter is inhibited when sib is present. This effect appears to be specific for PL because sib does not cause inhibition of PI-dependent int synthesis. lambda mutants that contain alterations in the site have been isolated. Sequence analyses of the mutations reveal single base changes, spanning 37 base pairs (bp) in the b region, some 240 bp beyond the int gene. Another mutant, hef13, which has a phenotype similar to that of sib, introduces a nucleotide change within the same 37-bp region. The sib and hef mutations cluster within a region of dyad symmetry. Regulation of int synthesis by sib occurs after transcription of the int gene. There is no difference in the rate of PL-promoted int mRNA synthesis in either sib+ or sib- phage infections, yet int mRNA is less stable in the sib+ infection. Because RNase III host mutants are defective in sib regulation, processing of the PL mRNA at sib by this endoribonuclease may cause int mRNA decay and decrease int synthesis.
Eukaryotic mRNA capping enzyme-guanylate covalent intermediate.
Authors: Venkatesan S , Moss B
Published: May 2006

Incubation of HeLa cell mRNA guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) with [alpha-32P]GTP and a divalent cation in the absence of an RNA acceptor results in the formation of a covalent enzyme-guanylate complex. The complex, after purification by phosphocellulose chromatography, can transfer its bound GMP moiety to pyrophosphate, regenerating GTP, or to the 5'-diphosphate end of poly(A), forming a cap structure G(5')pppA(pA)n. The GMP-polypeptide has a molecular weight of 65,000 and is stable to heating in the presence of sodium dodecyl sulfate. On the basis of the alkali-stable and acid-labile nature of the bond and its susceptibility to nucleophilic attack by hydroxylamine at low pH, the GMP-polypeptide linkage appears to be a phosphoamine bond. After digestion with trypsin, a single GMP-peptide was resolved by two dimensional electrophoresis and chromatography.
Association of cytosol hemoglobin with the membrane in intact erythrocytes.
Authors: Eisinger J , Flores J , Salhany J M
Published: May 2006

The problem of demonstrating hemoglobin binding to the erythrocyte membrane in intact cells was approached by observing the quenching of fluorescent membrane probes by hemoglobin as a function of pH. This quenching was studied by measuring fluorescence intensities and decay rates of membrane-bound donors by both right-angle and front-face fluorometry. The donors included 4,4'-bis(isothiocyano)-2,2'-stilbene disulfonate (DIDS) bound to the band 3 protein and 2-, 6-, and 12-(9-anthroyloxy(stearic acid (2-AS, 6-AS, and 12-AS) in the lipid portion of the membrane. The probe fluorescence is quenched progressively as the intracellular pH is decreased from 7.2 to 5.9 and does not depend on the oxygenation state. Since the fluorescence characteristic of the DIDS and x-AS probes in ghosts are independent of pH over this range, this quenching is due to the greater proximity of the donors and hemoglobin. Because hemoglobin binding to the band 3 protein in ghosts has previously been shown under conditions of low ionic strength and hemoglobin concentration, the present results were analyzed by a model in which a hemoglobin molecule is bound or in close proximity to a band 3 protein at pH 6. By using resonance energy transfer theory, we found the distance from DIDS to the bound hemoglobin to be approximately 4 A, which is within the range of distances measured between the DIDS binding site and the band 3 protein cytoplasmic sulfhydryl groups. Furthermore, the pH-dependent fluorescence quenching of 12-AS was stronger than that of 2-AS, showing that a cytoplasmic acceptor is involved and that the average distance between hemoglobin and these probes was greater than that for DIDS.
Purification and properties of a rat liver protein that specifically inhibits the proliferation of nonmalignant epithelial cells from rat liver.
Authors: McMahon J B , Farrelly J G , Iype P T
Published: May 2006

An inhibitor of cell proliferation was purified from rat liver by alcohol precipitation, ultrafiltration, and DEAE-cellulose chromatography. The hepatic proliferation inhibitor was shown to be pure by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, analytical isoelectric focusing, and high-performance liquid chromatography. The hepatic proliferation inhibitor was found to have a molecular weight of 26,000 and an isoelectric point of 4.65. This protein inhibited the proliferation of nonmalignant rat liver cells in culture, and removal of the protein reversed the inhibition produced by low doses. It exerted no effect on the proliferation of malignant rat liver cells.
Subunit treadmilling of microtubules or actin in the presence of cellular barriers: possible conversion of chemical free energy into mechanical work.
Authors: Hill T L , Kirschner M W
Published: May 2006

Free microtubule or actin filaments, along with the monomeric forms of the protein, hydrolyze GTP or ATP to produce a flux of subunits through the polymer. This flux, called treadmilling, produces no useful work. In the cell, however, these filaments are likely to be constrained between nucleating sites and other barriers that will limit polymer growth. We study here the effects of a small compression of the filaments resulting from polymerization against such barriers. If subunits can still exchange at the two ends, treadmilling will take place here as well. Under these conditions, the filament system can do useful work. The free energy of NTP hydrolysis can be used to transport materials, attached to the filament, against a resisting force. This process can in principle take place at high efficiency and bears a resemblance in a bioenergetic sense to the utilization of ATP free energy in muscle contraction. The same general principles apply to a polymer in which one end is anchored and one end is free.
Drosophila gene related to the major heat shock-induced gene is transcribed at normal temperatures and not induced by heat shock.
Authors: Ingolia T D , Craig E A
Published: May 2006

A gene related to the major heat shock-induced (hsp70) gene of Drosophila has been isolated from the sibling species D. melanogaster and D. simulans. This heat shock-cognate (hsc70) gene is present at cytological locus 70C. The primary sequence of approximately one-third of the protein-coding region has been compared with that of the hsp70 gene; 72% homology of the base sequence and 74% homology of the deduced amino acid sequence was found. In the codon specifying amino acid 66, the hsc70 gene contains an insertion of 1.7 kilobases; the hsp70 genes contain no intervening sequences. The sequence at the 5' and 3' junctions of the insertion is similar to that found in many intervening sequences. cDNA extension experiments indicate that the hsc70 gene is transcribed at normal temperatures in adult flies and that transcription is not enhanced by heat treatment.
Genetic variation in natural populations: problem of electrophoretically cryptic alleles.
Authors: Ayala F J
Published: May 2006

Electrophoretic studies have shown that the average frequency of heterozygous loci per individual is about 12% in Drosophila and other invertebrates and about 6% in vertebrates. It is estimated that only about two-thirds of all amino acid substitutions change net electric charge; hence, a large fraction of all genetic variation may be undetected by electrophoresis. Peptide mapping of 11 independent alleles coding for alcohol dehydrogenase in Drosophila melanogaster has uncovered one cryptic variant; thus, the frequency of electrophoretically cryptic variation is apparently low, about 9% in this sample. Nevertheless, with a simple model it is shown that this degree of cryptic variation, if it is typical of other loci, would substantially change our current estimates of genetic variation: the average heterozygosity would increase from about 12% to about 25% for invertebrates and from about 6% to 21% for vertebrates. A variety of techniques--including sequential electrophoresis and heat or urea denaturation--have been used by various investigators to detect electrophoretically cryptic variation. These techniques appear to be less effective than peptide mapping for detecting cryptic variation, but, like peptide mapping, they suggest that standard electrophoresis may detect most of the protein variation present in natural populations. The charge-state model of protein variation proposes that the "alleles" detected by electrophoresis are extremely diverse classes consisting of many electrophoretically cryptic alleles. The alcohol dehydrogenase peptide-mapping results are inconsistent with the charge-state model.
Anti-influenza response achieved by immunization with a synthetic conjugate.
Authors: Müller G M , Shapira M , Arnon R
Published: May 2006

The peptide corresponding to sequence 91--108 of the hemagglutinin of type A H3N2 influenza virus has been synthesized by the solid-phase peptide synthesis method and covalently attached to several macromolecular carriers. The conjugate with tetanus toxoid was used for immunization of rabbits and mice. The immunoglobulin fraction of the rabbit antiserum showed the presence and antipeptide antibodies by both agar gel diffusion and radioimmunoassay. In the latter assay, the antibodies showed marked crossreactivity with the intact virus of the A/Texas/77 strain. The antibodies were also capable of inhibiting the hemagglutination of chicken erythrocytes by the virus; the highest hemagglutination inhibition titer (1:32) was achieved with a serum-resistant strain of A/Texas/77. When the in vitro virus plaque formation assay was used with monolayers of Madin--Darby canine kidney (MDCK) cells, the number of plaques was reduced on interaction with the immunoglobulin fraction of the antiserum, which was effective up to a dilution of 1:32. Preliminary results indicate that C3H/DiSn mice immunized with the peptide--tetanus toxoid conjugate are partially protected against a further challenge with A/Texas mouse-adapted influenza virus. The results are thus indicative of the efficacy of the synthetic material in eliciting anti-influenza immune response.
Hepatitis B virus vaccine: identification of HBsAg/a and HBsAg/d but not HBsAg/y subtype antigenic determinants on a synthetic immunogenic peptide.
Authors: Prince A M , Ikram H , Hopp T P
Published: May 2006

On the basis of theoretical considerations, a peptide (H peptide) was synthesized by Hopp and Woods [Hopp, T. P. & Woods, K. R. (1981) Proc. Natl. Acad. Sci. USA 78, 3824---3828]. This peptide contains a sequence of six amino acids postulated to represent the major epitope, or antibody-combining site, of hepatitis B virus surface antigen (HBsAg). We have used passive hemagglutination inhibition with monospecific antibodies against the a, d, and y subdeterminants of this antigen and against human serum albumin to investigate the antigenic specificities on this peptide, and we have found it to contain the HBsAg/a and HBsAg/d but not HBsAg/y or human serum albumin subdeterminants. When the peptide was conjugated onto human erythrocytes and injected into mice, it induced the formation of anti-HBsAg with and without the use of Freund's adjuvant. If anti-HBsAg/a confers immunity to infection with hepatitis B virus, as is generally thought, these findings may permit the development of a synthetic vaccine lacking all unnecessary antigenic determinants.
Behaviorally conditioned suppression of a graft-versus-host response.
Authors: Bovbjerg D , Ader R , Cohen N
Published: May 2006

Cyclophosphamide (CY), previously used to condition suppression of humoral immune responses, was used to condition suppression of a graft-versus-host response (GvHR). Female (Lewis x Brown Norway) F1 rats were conditioned by pairing consumption of a saccharin solution with an intraperitoneal injection of CY at 50 mg/kg of body weight 48 days before immunization. On day 0, all animals were injected with a suspension of splenic leukocytes (2 x 10(7) cells per footpad) obtained from female Lewis donors. The regional GvHR was assessed on day 5 by weighing popliteal nodes. Conditioned animals given a single low-dose injection of CY and reexposed to conditioned stimuli had lymph node weights significantly lower than control groups and did not differ from animals given three injections of CY during the ongoing GvHR. The results suggest that conditioned immunosuppression, previously demonstrated in thymus-dependent and thymus-independent humoral immune responses, also affects the popliteal GvHR, a cellular immune response.
Transcription terminates at lambda tR1 in three clusters.
Authors: Lau L F , Roberts J W , Wu R
Published: May 2006

Transcription of the rightward operon of bacteriophage lambda terminates in the presence of rho factor at a region known as tR1, located downstream of the structural gene for the lytic repressor cro. We demonstrate by nuclease Sl mapping that transcription termination at lambda tR1 occurs in vitro over a stretch of 60 nucleotides. End points of transcripts are clustered in three distinct regions, which we refer to as termination sites I, II, and III. Termination at site I is inhibited by L factor, whereas termination at sites II and III is not affected by L factor. The sensitivities of these sites to rho factor are in the order III greater than II greater than I. The cin-lcnc-l mutations abolish termination at site II but not at sites I and III; this result may explain the failure of these mutations to alleviate the phage requirement for N function for growth. Although possible stem-and-loop structures in the RNA transcript immediately upstream of each of the three termination sites can be found, no consistent correlation exists between the strengths of these stem-and-loop structures and the termination efficiencies of their respective sites.
Relationship between the total size of exons and introns in protein-coding genes of higher eukaryotes.
Authors: Naora H , Deacon N J
Published: May 2006

We have attempted to ascertain the correlation between the genetic information content in the exons and the surrounding intron sequences with regard to their spatial arrangement within a gene. A comparison is made of the sizes, taken from recent publications, of exons and introns of approximately equal to 80 different protein-coding chromosomal genes, mostly from higher eukaryotes. The exons of these genes do not show very marked variation in size and can be classified into three major discrete and two minor additional size groups, whereas individual introns vary considerably in size within and between genes. Notwithstanding, the overall length of all introns present within a given gene is a function of the total size, mostly corresponding to the total genetic information content, of the exons. Three cases that violate this exon-size dependency of introns are genes coding for (i) histone H1, feather keratin, and interferons, (ii) tubulin and actin, and (iii) silk fibroin. The exons of these genes are larger than 0.7 kilobase pair in total size and the genes show a strong sequence homogeneity among the repetitious family members or internal repeats of coding sequences within the gene. We propose that conservation of sequences, which is required by the family members, internal repeats, or the entire gene, would actually motivate the removal of introns.
Control of ColE1 DNA replication: the rop gene product negatively affects transcription from the replication primer promoter.
Authors: Cesareni G , Muesing M A , Polisky B
Published: May 2006

A 600-base-pair region essential for ColE1 and pMBl plasmid replication contains two promoters responsible for the synthesis of two RNA molecules central to copy number control. One promoter directs synthesis of the primer RNA precursor. The second promoter directs the synthesis of a small RNA molecule, RNAl, which acts in trans to inhibit processing of the RNA primer precursor. We have fused each promoter to the beta-galactosidase structural gene contained in a lambda phage. Expression of the RNAl promoter in lysogens is not influenced by the presence of wild-type pMBl or ColEl plasmids residing in the cell. Transcription from the RNA primer promoter, however, is repressed by the product of a trans-acting plasmid gene product, which we have designated rop (for repressor of primer). The rop gene maps downstream from the replication origin in a region that encodes a polypeptide of 63 amino acids whose sequence is completely conserved in pMBl and ColE1. We propose that this polypeptide is the rop gene product and that it regulates plasmid DNA replication by modulating the initiation of transcription of the primer RNA precursor.
Short direct repeats flank the T-DNA on a nopaline Ti plasmid
Authors: Yadav Narendra S. , Vanderleyden Jos , Bennett Donald R. , Barnes Wayne M. , Chilton Mary-Dell
Published: May 2006

Crown gall disease results from the insertion of a segment of the
Agrobacterium
Ti plasmid, called T-DNA, into host plant nuclear DNA. We have subjected to sequence analysis the border regions of pTi T37 (ends of T-DNA) and one left T-DNA/plant DNA border fragment isolated from BT37 tobacco teratoma by molecular cloning. These sequence studies, taken together with published sequence of a right T-DNA/plant DNA border fragment, allowed us to identify the positions of left and right borders at the DNA sequence level. Comparison of left and right border regions of the Ti plasmid revealed a “core” direct repeat of 13 of 14 bases (12 contiguous) precisely at the borders of T-DNA. An extended repeat of 21 of 25 bases overlaps this core repeat. T-DNA on the Ti plasmid exhibits no longer direct or inverted repeats in the border regions, based on Southern hybridization studies. The physical structure of T-DNA differs from that of known prokaryotic and eukaryotic transposable elements but bears a structural resemblance to the prophage of bacteriophage λ.
Cloning and characterization of different human sequences related to the onc gene (v-myc) of avian myelocytomatosis virus (MC29).
Authors: Dalla-Favera R , Gelmann E P , Martinotti S , Franchini G , Papas T S , Gallo R C , Wong-Staal F
Published: May 2006

We have studied the genomic organization of human cellular sequences (c-myc) homologous to the transforming gene (v-myc) of avian myelocytomatosis virus (MC29). Southern blotting experiments using v-myc probes showed that several fragments of the human genome contain sequences related to the central part of v-myc but only few of them are homologous to the 3' portion of the viral gene. Several recombinant phages which represent different regions of the genome containing c-myc-related sequences were isolated from a human DNA library. Two clones (lambda-LMC-12 and -41) overlap over approximately 17 kilobases of DNA where a sequence homologous to that of the entire v-myc is present. Restriction mapping experiments and heteroduplex analysis show that c-myc sequences of this locus are interrupted by one intron, suggesting that lambda-LMC-12 and -41 contain the complete functional c-myc gene. Three other clones (lambda-LMC-3, -4, and -26) do not overlap and contain sequences related to only approximately 0.3 kilobase of v-myc but lack 5' and 3' portions of the gene. These sequences are not interrupted by introns and are more divergent from v-myc than is the complete gene, suggesting that they may represent either pseudogenes or parts of distantly related genes.
alpha-Amylase biosynthesis: evidence for temporal sequence of NH2-terminal peptide cleavage and protein glycosylation.
Authors: Miyata S , Akazawa T
Published: May 2006

During the biosynthesis of secretory proteins, an NH2-terminal peptide, referred to as a signal peptide, is cotranslationally cleaved off after the protein enters the cisternal space of the endoplasmic reticulum. It also has been reported that the core glycosylation reaction of some secretory and viral membrane glycoproteins occurs as a cotranslational event. However, despite a huge amount of work, no decisive answer has been given as to the temporal sequence of proteolytic cleavage of the signal peptide and glycosylation on the polysomes for any secretory or membrane glycoprotein. We show here that proteolytically processed and already glycosylated chains of rice seed alpha-amylase exist on the polysomes; furthermore, our results provide direct evidence that glycosylation is preceded by proteolytic processing during the biosynthesis of the alpha-amylase molecule.
DNA sequence associated with chromosome translocations in mouse plasmacytomas.
Authors: Harris L J , D'Eustachio P , Ruddle F H , Marcu K B
Published: May 2006

A DNA sequence that generates aberrantly rearranged immunoglobulin heavy chain constant region genes in murine plasmacytomas is shown to participate in a chromosome translocation. We have previously termed this DNA sequence NIARD for non-immunoglobulin-associated rearranging DNA. NIARD rearrangements were found frequently in murine plasmacytomas but were not detected in normal lymphocytes. These rearrangements occasionally involve the switch region of the C alpha gene. In this study, DNA samples obtained from mouse-Chinese hamster somatic cell hybrid lines were digested with various restriction endonucleases and analyzed by the Southern transfer technique with a NIARD hybridization probe. These experiments show that NIARD resides on chromosome 15 in the mouse germ line. Since NIARD is found adjacent to the C alpha gene (located on chromosome 12) in some plasmacytomas, it is apparent that a translocation involving these two chromosomes has occurred. We have proposed a rcpT(12;15) model to explain our data. The implications of NIARD rearrangements for malignant transformation are discussed.
Expression of Ia-like antigens by human vascular endothelial cells is inducible in vitro: demonstration by monoclonal antibody binding and immunoprecipitation.
Authors: Pober J S , Gimbrone M A
Published: May 2006

The expression of Ia-like antigens by cultured human endothelial cells has been investigated by means of monoclonal antibody binding to intact cells and by immunoprecipitation of radioiodinated membrane proteins. Primary growing and confluent cultures of human umbilical vein endothelium express little, if any, detectable Ia-like antigens under standard culture conditions. However, treatment of primary cultures with the lectin phytohemagglutinin induces the expression of Ia-like antigens. This action of the lectin uniformly affects all the endothelial cells in a culture, does not depend on cell division, and is associated with a cell shape change. The data presented in this report provide unequivocal serological and biochemical demonstration of Ia-like antigens on human vascular endothelial cells. The fact that the expression of Ia-like antigens by endothelium can be induced may have important implications for organ transplantation and for regulation of the immunological response.
Affinity labeling of a transforming growth factor receptor that does not interact with epidermal growth factor.
Authors: Massague J , Czech M P , Iwata K , DeLarco J E , Todaro G J
Published: May 2006

Membrane components that interact with epidermal growth factor (EGF) and transforming growth factors (TGFs) have been identified by covalent crosslinking to their respective 125I-labeled ligands. Under appropriate conditions, disuccinimidyl suberate or hydroxysuccinimidyl p-azidobenzoate cross-link receptor-bound 125I-labeled EGF to a 140- to 170-kilodalton (kDal) receptor species in membranes from both A431 human carcinoma cells and normal rat kidney cells. 125I-Labeled sarcoma growth factor (SGF), a TGF from virally transformed mouse 3T3 cells, also can be affinity-crosslinked to the 140- to 170-kDal EGF receptor species in membranes from A431 and rat kidney cells. The labeling of this receptor is inhibited when either excess unlabeled EGF or SGF is present during incubation of membranes with either 125I-labeled EGF or 125I-labeled SGF. In contrast, a second receptor species of 60 kDal is affinity-labeled with 125I-labeled SGF but not with 125I-labeled EGF in membranes from both A431 and rat kidney cells. SGF and a TGF from virally transformed rat embryo cells inhibit the labeling of the 60-kDal species when present in excess during incubation of membranes with 125I-labeled SGF, whereas EGF is completely ineffective in inhibiting the labeling of this receptor. The data suggest that a specific 60-kDal receptor that displays high affinity for TGFs but not for EGF may mediate induction of the transformed phenotype. In addition, SGF and other TGFs interact with the 140- to 170-kDal EGF receptor that appears to mediate normal cell growth effects.
Exon cloning: immunoenzymatic identification of exons of the chicken lysozyme gene.
Authors: Rüther U , Koenen M , Sippel A E , Müller-Hill B
Published: May 2006

A 10-kilobase DNA fragment containing exons 1 and 2 of the chicken lysozyme gene has been randomly cleaved with DNase I. After tailing and cloning into the plasmid pUK230, Lac+ colonies were selected. Colonies harboring expressed fragments of the exons could be detected by an immunoenzymatic assay using antibodies against lysozyme. The smallest fragment coded for 10 amino acids and the largest coded for almost all residues of exon 2. These results suggest that any gene of any genome cloned in this way can be detected if antibodies against the gene product are available.