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PubMed1989

Steroid C-20 oxidoreductase activity of duck intestinal mucosa: the interrelations of the enzymatic activity with steroid binding.

The binding of corticosterone and aldosterone to domestic duck (Anas platyrhynchos)-dispersed colonic mucosal cells at 37 degrees was investigated. It was found that in contrast to experiments using cell-free intestinal preparations, corticosterone was extensively metabolized and it was the metabolite, not the native steroid that became receptor bound and all the bound ligand was in the nuclear fraction. The metabolite turned out to be identical with 4-pregnene-11 beta,20 beta, 21-triol-3-one (20 beta-dihydrocorticosterone, 20 beta-DHB). Binding experiments with [3H]corticosterone yielded the following kinetic parameters: Kd = 87.6 nM, Nmax = 337,900 sites/cell. When synthetic [3H]20 beta-DHB was used as the ligand a curvilinear-binding isotherm was obtained. This could be resolved into a high affinity-low capacity (HA) and a low affinity-high capacity (LA) component with the following binding parameters: Kd,HA = 91 nM, Nmax,HA = 130,800 sites/cell; Kd,LA = 5.4 x 10(-6) M, Nmax, LA = 3.7 x 10(6) sites/cell. Binding of the metabolite to cell-free preparations, at 0 degree, gave the following results: for cytosol, linear-binding isotherm, Kd = 14.0 nM, Nmax = 26.5 fmol/mg protein; and for crude nuclei, curvilinear-binding isotherm, Kd,HA = 45.0 nM, Nmax, HA = 5.33 pmol/mg DNA; Kd,LA = 2.2 x 10(-6) M, Nmax,LA = 286.6 pmol/mg DNA. [3H]Aldosterone was also bound by the dispersed whole cells and again, this binding was only nuclear (Kd = 9.3 nM, Nmax = 10,042 sites/cell). The bound ligand was unchanged aldosterone. Competition experiments have shown that aldosterone did not compete with 20 beta-DHB for binding sites and vice versa. The intracellular 20 beta-hydroxysteroid oxidoreductase responsible for the transformation of corticosterone was found mostly in the cytoplasm. Kinetic studies with the enzyme yielded classical Michaelis-Menten kinetics (Km = 15.7 microM, Vmax = 2.6 nmol/min/mg protein). The enzyme had an apparent Mr of 35 kDa and a Rs of 25.5 A. It is believed that our results might explain the binding of aldosterone to mineralocorticoid-binding sites in the presence of overwhelming concentrations of corticosterone and that experiments with cell-free tissue preparations, performed at 0 degree, do not reflect the true cellular-binding events.

20-Hydroxysteroid Dehydrogenases↗
PubMed2006

The atlA operon of Streptococcus mutans: role in autolysin maturation and cell surface biogenesis.

The Smu0630 protein (AtlA) was recently shown to be involved in cell separation, biofilm formation, and autolysis. Here, transcriptional studies revealed that atlA is part of a multigene operon under the control of at least three promoters. The morphology and biofilm-forming capacity of a nonpolar altA mutant could be restored to that of the wild-type strain by adding purified AtlA protein to the medium. A series of truncated derivatives of AtlA revealed that full activity required the C terminus and repeat regions. AtlA was cell associated and readily extractable from with sodium dodecyl sulfate. Of particular interest, the surface protein profile of AtlA-deficient strains was dramatically altered compared to the wild-type strain, as was the nature of the association of the multifunctional adhesin P1 with the cell wall. In addition, AtlA-deficient strains failed to develop competence as effectively as the parental strain. Mutation of thmA, which can be cotranscribed with atlA and encodes a putative pore-forming protein, resulted in a phenotype very similar to that of the AtlA-deficient strain. ThmA was also shown to be required for efficient processing of AtlA to its mature form, and treatment of the thmA mutant strain with full-length AtlA protein did not restore normal cell separation and biofilm formation. The effects of mutating other genes in the operon on cell division, biofilm formation, or AtlA biogenesis were not as profound. This study reveals that AtlA is a surface-associated protein that plays a critical role in the network connecting cell surface biogenesis, biofilm formation, genetic competence, and autolysis.

Bacterial Proteins↗
PubMed1981

Haemophilus influenzae polypeptides involved in deoxyribonucleic acid uptake detected by cellular surface protein iodination.

Polypeptides that appear to be involved in competence development and deoxyribonucleic acid (DNA) uptake by Haemophilus influenzae were detected with a surface-specific iodinating reagent 1,3,4,6,-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril. As shown on electrophoretograms, a number of polypeptides became sensitive to 125I protein labeling with the ability of these cells to bind DNA. Of these polypeptides, nine were reduced in their ability to be labeled (ral polypeptides) extensively after the incubation of competent cells with homologous, but not with heterologous, DNA. Iodination of many of these ral polypeptides was reduced in competence-deficient mutants compared with wild-type competent cells. One 125I-labeled polypeptide corresponding to a molecular weight of 29,000 was present at reduced levels in mutants reduced in the ability to bind DNA. Our results suggest that the 29,000-molecular-weight polypeptide corresponds with the ability of H. influenzae to take up DNA and that a complex of proteins is involved in DNA uptake and transformation.

Bacterial Proteins↗
PubMed1975

Frequency of infection and efficiency of transfection of Rhizobium meliloti cells and spheroplasts.

Competent cultures of Rhizobium meliloti cells and spheroplasts obtained by various methods were infected with DNA of phage 1A. The Frequency of infection among the cells and spheroplasts was 2 X 10(-8)-5 X 10(-10). The efficiency of transfection calculated from the ratio of plaque forming units to infective DNA molecule of phage 1A was 5 X 10(-8) to 10(-10). Frequency of infection and efficiency of transfection among the competent cells were by one order of magnitude higher in the case of the spheroplasts. The use of various media did not noticeably alter the efficiency of transfection.

Bacteriophages↗
PubMed1995

Transformation of naturally competent Streptococcus mutans with replicative and non-replicative Tn916-containing plasmids: implications for a mechanism of transposition.

Based on the observations reported here and what is known concerning transformation of naturally competent strains of S. mutans and other streptococcal species such as S. gordonii, we propose the model shown in Figure 2. The Tn916-intermediate transforms S. mutans as originally proposed for B. subtilis by Scott and coworkers [8]. It is not clear in either system (B. subtilis or S. mutans) whether the Tn916 intermediate enters the cell as ds-DNA or ss-DNA. Because it is likely that transformation of B. subtilis via formation of protoplasts involves a mechanism quite different from natural transformation in S. mutans, it would be unwise to extrapolate findings from their studies. If Tn916 enters S. mutans in a manner similar to plasmid or chromosomal DNA, we would assume that Tn916 binds to a cell receptor and as one strand enters, the other is degraded [9]. This leaves open the question of whether Tn916 recircularizes as ds-DNA before it inserts into the chromosome or whether it remains as ss-DNA, if, indeed, it enters as ss-DNA. The transformation efficiency for the Tn916 intermediate (approximately 10(-7) precluded kinetics studies such as those performed with pAM118. Poyart-Salmeron and coworkers [11] however, described a model in which Tn1545 inserts into the target site as a ds-DNA circular molecule, similar to that seen with lambda phage. Perhaps the most interesting finding presented here is that the predominant mechanism of insertion of Tn916 into the chromosome of the recipient occurs after Tn916 enters the cell. The replicative plasmid pAM118 evidently forms by two-hit kinetics followed by intracellular excision and transposition of Tn916. The helper-rescue experiment shows that in this system, the formation of Tcr transformants, and hence the integration of Tn916, was a function of the transformation efficacy of plasmid pAM118. Since intracellular excision of Tn916 probably follows the re-formation of the plasmid pAM118, the rate-limiting step in this system would be the formation of the transient, intracellular plasmid pAM118. (The transient white colony phenotype probably denotes the slower growth rate of transformants that acquire the large replicative plasmid pAM118). Our findings demonstrate that a practical way of promoting Tn916 insertions into chromosomal DNA for the purpose of obtaining mutations is to use a helper-rescue system. Our model supports the concept that the majority of Tn916 inserts arise from a mechanism similar to 'zygotic induction' as proposed for S. sanguis (gordonii) [1]. However, the frequencies for the co-establishment of the replicative plasmid (Emr) and Tn916 inserts (Tcr) in their paper (10(-6)) differ from our observations for S. mutans. We found nearly 100% of Tcr white colonies to be Emr whereas in S. gordonii, only a fraction (approximately 1%) exhibited the TcrEmr phenotype. If both phenotypes arose independently, the frequency of the TcrEmr phenotype would be 10(-8) rather than 10(-6) survivors/recipients as observed. It was surmised that both Tcr and Emr transformants arise dependently [1] where both the Tn916 intermediate and pAM118 contribute to the formation of Tn916 inserts. We conclude from their data, however, that most Tcr arose from the Tn916-intermediate formed in the donor, in agreement with the explanation of these data by Scott [12]. Support for the contention that the Tcr arose in S. gordonii from transformation by the Tn916-intermediate as in S. mutans (yellows), and not from intracellular excision from pAM118, comes from the observation that so few Tcr are Ems and because pAM150 (rep-) yields Tcr at the same frequency as the rep+ pAM118 in their experiments. In summary, the Tn916 intermediate is capable of transforming S. mutans. In contrast to the hypothesis of Scott [12], however, the Tn916 intermediate is not the only form involved in the transformat++t

Chromosomes, Bacterial↗
PubMed1996

Identification of the streptococcal competence-pheromone receptor.

Competence for genetic transformation in certain species of streptococci has been known for many years to be induced by a secreted protease-sensitive pheromone, referred to as the competence factor or activator, which acts as a quorum-sensing signal to co-ordinate expression of late competence genes. We recently reported identification of the pheromone of Streptococcus pneumoniae strain Rx as a small unmodified peptide, which was termed competence-stimulating peptide (CSP). By identifying the gene (comC) encoding the Rx CSP we were able to show that it is synthesized as a precursor peptide containing an N-terminal double-glycine type leader. In the present work, we describe two alleles of the corresponding gene from Streptococcus gordonii strains Challis and NCTC 7865, which are strains with distinct competence pheromones and corresponding specific pheromone reactivities. In addition, the nucleic acid sequences of two genes located downstream of comC were determined; interestingly, these genes encode a two-component signal transduction system. We therefore speculated that their products, a histidine kinase (ComD) and its cognate response regulator (ComE), act downstream of the CSP in competence regulation. By tracing the CSP specificity of the competence response in these strains to strain-specific alleles of comD, we obtained evidence demonstrating that the histidine kinase ComD is the competence-pheromone receptor.

Alleles↗
PubMed2006

Inhibition of transforming growth factor-beta1 signaling attenuates ataxia telangiectasia mutated activity in response to genotoxic stress.

Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor beta (TGFbeta)-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGFbeta inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgfbeta1 null murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGFbeta type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced gammaH2AX radiation-induced foci; and increased radiosensitivity compared with TGFbeta competent cells. We determined that loss of TGFbeta signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGFbeta restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgfbeta1, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGFbeta may be used to advantage in cancer therapy.

Activin Receptors, Type I↗
PubMed2002

Genetic analysis of high-risk e6 in episomal maintenance of human papillomavirus genomes in primary human keratinocytes.

Papillomaviruses possess small DNA genomes that encode five early (E) proteins. Transient DNA replication requires activities of the E1 and E2 proteins and a DNA segment containing their binding sites. The E6 and E7 proteins of cancer-associated human papillomavirus (HPV) transform cells in culture. Recent reports have shown that E6 and E7 are necessary for episomal maintenance of HPV in primary keratinocytes. The functions of E6 necessary for viral replication have not been determined, and to address this question we used a recently developed transfection system based on HPV31. To utilize a series of HPV16 E6 mutations, HPV31 E6 was replaced by its HPV16 counterpart. This chimeric genome was competent for both transient and stable replication in keratinocytes. Four HPV16 E6 mutations that do not stimulate p53 degradation were unable to support stable viral replication, suggesting this activity may be necessary for episomal maintenance. E7 has also been shown to be essential for episomal maintenance of the HPV31 genome. A point mutation in the Rb binding motif of HPV E7 has been reported to render HPV31 unable to stably replicate. Interestingly, HPV31 genomes harboring two of the three p53 degradation-defective E6 mutations combined with this E7 mutation were maintained as replicating episomes. These findings imply that the balance between E6 and E7 functions in infected cells is critical for episomal maintenance of high-risk HPV genomes. This model will be useful to dissect the activities of E6 and E7 necessary for viral DNA replication.

Amino Acid Sequence↗
PubMed1991

Expression of mouse cytochrome P450IA1 cDNA in repair-deficient and repair-proficient CHO cells.

Recombinant DNA techniques have been used to develop Chinese hamster ovary cell lines for studying chemically induced genotoxicity. These cell lines express a specific cytochrome P450 isozyme responsible for the metabolism of polycyclic aromatic hydrocarbons and exhibit defined differences in DNA repair capacity. A bacterial gene (neo) conferring resistance to gentamicin was inserted into the pcD expression vector containing the mouse cytochrome P1450 (P450IA1) cDNA to facilitate the selection of transformed cells. This plasmid was introduced into the nucleotide-excision-repair-deficient UV5 cell line by electroporation. Transformed clonal isolates expressing the P1450 cDNA were identified by differential cytotoxicity assays using benzo[a]pyrene (B[a]P). One such clone, termed UV5P1, was mutagenized with ethyl methanesulfonate and selected for resistance to killing by UV radiation to derive a repair-competent clone that expresses similar P1450 activity to that of the parental cell line. Two repair-competent clones were selected and called 5P1R1 and 5P1R3. The resulting cell lines (UV5P1, 5P1R1, and 5P1R3) expressed arylhydrocarbon hydroxylase activity. UV5P1 and 5P1R3 were compared in terms of cytotoxicity and mutagenicity after exposure to B[a]P. Induced mutations were measured at the adenine phosphoribosyltransferase (aprt) and hypoxanthine guanine phosphoribosyltransferase (hprt) loci. Repair-deficient UV5P1 cells were killed by B[a]P at concentrations below 0.1 microM, while the repair-proficient 5P1R3 cells showed no toxicity up to 60 microM. Mutation induction at both loci was also much more efficient in UV5P1 cells. These new cell lines provide a more sensitive system that can be used in a battery of assays to evaluate the genotoxicity of chemicals requiring P450IA1 metabolic activation and to assess the role of DNA repair in modulating the biological effects of DNA damage.

Animals↗
PubMed1998

The M4M5 cytoplasmic loop of the Na,K-ATPase, overexpressed in Escherichia coli, binds nucleoside triphosphates with the same selectivity as the intact native protein.

Escherichia coli was used to overexpress the large cytoplasmic loop of the rat Na,K-ATPase. A 1260-base DNA segment encoding Lys354-Lys774 of the rat alpha1-subunit was constructed via polymerase chain reaction. The polymerase chain reaction product was successfully subcloned into the expression vector pET-28 (Novagen), which produces an N-terminal 6-histidine-tagged fusion protein. The pET-28 vector containing rat alpha-loop, i.e. pAN, was used to transform calcium-competent E. coli BL21(DE3) cells, and positive clones were selected by kanamycin resistance. Bacterial cultures were grown, and protein synthesis was induced with isopropyl beta-D-thiogalactoside. Cells were harvested and lysed, revealing production of the His-tagged fusion protein ( approximately 46 kDa). The fusion protein was affinity-purified from other soluble cellular proteins via a Ni-NTA column, which routinely yielded approximately 20 mg of soluble His6-alpha-loop/L cell culture. The His6-alpha-loop retained significant native structure, as evidenced by the ability of ATP and ADP (but not AMP, CTP, GTP, or UTP) to protect against chemical modification by either fluorescein isothiocyanate or maleimidylanilinonapthalene sulfonic acid. More specifically, circular dichroism spectroscopy was used to estimate the secondary structure of the His6 loop, revealing an ordered folding composed of 23% alpha-helix, 23% antiparallel beta-sheet, 4% parallel beta-sheet, 19% beta-turn, and 32% random coil. The 6-histidine loop bound the fluorescent ATP analog trinitrophenyl-ATP with high affinity, as determined by measuring the fluorescence changes associated with binding. Affinities for ATP ( approximately 350 microM) and ADP ( approximately 550 microM) were determined by their ability to compete with and displace 2',3'-O-[2,4,6,-trinitrophenyl]-ATP. These nucleotide affinities are similar to those observed for the E2 conformation of the intact Na,K-ATPase.

Adenosine Diphosphate↗
PubMed1997

Arginine-, hypoxanthine-, uracil-requiring isolates of Neisseria gonorrhoeae are a clonal lineage with a non-clonal population.

Multilocus enzyme electrophoresis has shown that a collection of 101 arginine-, hypoxanthine-, uracil-requiring (AHU-) isolates of Neisseria gonorrhoeae, recovered over a 39 year period from the UK and Denmark, were of a single electrophoretic type (91% of strains), or differed from the predominant electrophoretic type at only a single locus. The striking uniformity of the AHU-isolates, and the correlation between auxotype, serovar and overall genetic background, contrasts with previous studies of gonococcal populations (that included very few AHU-strains), and a small sample of non-AHU-isolates studied here, which demonstrated a non-clonal population structure and a lack of association between auxotype, serovar and genetic background. There was no marked difference in the ability of AHU-isolates to be transformed with their own DNA, or with DNA from gonococci of other auxotypes, and the relative genetic stability of AHU-isolates does not appear to be due to a defect in their ability to be transformed. An alternative possibility is that AHU-gonococci recombine with other lineages, but that the resulting recombinants are not maintained in the population. This would occur, for example, if AHU-gonococci competed poorly in mixed infections, within which effective recombination between lineages occurs, and are usually only transmitted from individuals who are singly infected with an AHU-strain. The association between AHU-gonococci and asymptomatic infections may lead to an increased rate of transmission of these strains which under this scenario would be needed to prevent them from being lost from the population.

Arginine↗
PubMed1996

Regulation of competence for genetic transformation in Streptococcus pneumoniae by an auto-induced peptide pheromone and a two-component regulatory system.

The regulation of competence for genetic transformation in Streptococcus pneumoniae depends on a quorum-sensing system, but the only molecular elements of the system whose specific role have been identified are an extracellular peptide signal and an ABC-transporter required for its export. Here we show that transcription of comC, the gene encoding a predicted 41-residue precursor peptide that is thought to be processed and secreted as the 17-residue mature competence activator, increased approximately 40-fold above its basal level of expression in response to exogenous synthetic activator, consistent with earlier experiments indicating that the activator acts autocatalytically. We also describe two new genes, comD and comE, that encode members of histidine protein kinase and response-regulator families and are linked to comC. Disruption of comE abolished both response to synthetic activator peptide and endogenous competence induction.

Amino Acid Sequence↗
PubMed2002

Human papillomavirus oncoprotein E6 inactivates the transcriptional coactivator human ADA3.

High-risk human papillomaviruses (HPVs) are associated with carcinomas of the cervix and other genital tumors. The HPV oncoprotein E6 is essential for oncogenic transformation. We identify here hADA3, human homologue of the yeast transcriptional coactivator yADA3, as a novel E6-interacting protein and a target of E6-induced degradation. hADA3 binds selectively to the high-risk HPV E6 proteins and only to immortalization-competent E6 mutants. hADA3 functions as a coactivator for p53-mediated transactivation by stabilizing p53 protein. Notably, three immortalizing E6 mutants that do not induce direct p53 degradation but do interact with hADA3 induced the abrogation of p53-mediated transactivation and G(1) cell cycle arrest after DNA damage, comparable to wild-type E6. These findings reveal a novel strategy of HPV E6-induced loss of p53 function that is independent of direct p53 degradation. Given the likely role of the evolutionarily conserved hADA3 in multiple coactivator complexes, inactivation of its function may allow E6 to perturb numerous cellular pathways during HPV oncogenesis.

Antineoplastic Agents↗
PubMed1990

Transcriptional regulation of comC: evidence for a competence-specific transcription factor in Bacillus subtilis.

comC specifies a protein product that is required for genetic competence in Bacillus subtilis. The probable transcriptional start site of comC has been localized by high-resolution primer extension analysis and shown to be preceded by an appropriately positioned sequence that resembles the consensus promoter for the sigma A form of RNA polymerase. Low-resolution S1 nuclease transcription mapping was used to identify the comC terminator, which is located near a palindromic element recognizable in the DNA sequence. Deletion analysis of the sequence upstream from the likely promoter identified a region required in cis for the expression of comC. An overlapping, and possibly identical, sequence was shown to inhibit the expression of competence and of several late competence genes, when present in multiple copies. This was interpreted as due to the titration of a positively acting competence transcription factor (CTF) by multiple copies of the promoter-bearing fragment. In crude lysates of B. subtilis grown to competence, a DNA-binding activity that appeared to be specific for the comC promoter fragment was detected by gel retardation assays. This activity, postulated to be due to CTF, was detected only following growth in competence medium, only in the stationary phase of growth, and was dependent on the expression of ComA, a known competence-regulatory factor. In the presence of the mecA42 mutation, the ComA requirement for CTF activity was bypassed, and CTF activity could be detected in lysates prepared from a strain grown in complex medium. This behavior suggested that either the expression or the activation of CTF was regulated in a competence-specific manner. Comparison of the putative CTF-binding site defined by deletion analysis with a similarly positioned sequence upstream from the start site of the late competence gene comG revealed that both sequences contained palindromes, with 5 of 6 identical base pairs in each arm. It is suggested that these palindromic sequences comprise recognition elements for CTF binding and that CTF binding must occur for the appropriate expression of late competence genes.

Bacillus subtilis↗
PubMed1990

Mutational analysis of v-rel, the oncogene of reticuloendotheliosis virus strain T.

Reticuloendotheliosis virus strain T (Rev-T) is a highly oncogenic retrovirus that induces a lethal lymphoma in young galliform birds and also transforms and immortalizes avian lymphoid cells in vitro. Rev-T presumably arose when a reticuloendotheliosis virus (Rev) incorporated a portion of the turkey c-rel gene into its genome, forming the v-rel oncogene. The predicted v-rel protein (p59v-rel) has 14 amino acid substitutions and 3 amino acid deletions relative to the predicted turkey c-rel protein. The 5' Rev env-derived amino-terminus of p59v-rel also has amino acid substitutions in codons 3, 6, and 9 relative to wild-type Rev-A env. To distinguish the critical alterations from the neutral ones, we made a mutation that resulted in a substitution of the 5' Rev env-derived amino-terminus by a single methionine, as well as several back-mutations, in the context of an otherwise wild-type v-rel and tested the transforming, immortalizing, and tumorigenic properties of the resulting proteins. All proteins tested retained the transforming, immortalizing, and tumorigenic functions of v-rel. Absence of the 5' Rev env-derived amino-terminus, as well as back-mutations of six of the amino acid changes present in the rel-derived amino-terminal and central regions of v-rel, reduced transformation efficiency. The mutations present in codons 3, 6, and 9 of p59v-rel showed complicated interactions in their activating effects on transformation efficiency. We also showed that loss of the immortalizing function did not abolish tumorigenesis by v-rel. We further showed that the primary transduction event involving the turkey c-rel gene could have occurred with a replication-competent Rev having v-rel-specific mutations in codons 3, 6, and 9 of the env gene.

Amino Acid Sequence↗
PubMed1988

Capturing nuclear sequence-specific DNA-binding proteins by using simian virus 40-derived minichromosomes.

We have used recombinant simian virus 40 (SV40) minichromosomes to retrieve sequence-specific DNA-binding proteins derived from the cell nucleus of COS-7 cells. We showed that the transcription factors AP-1 and Sp1 are stably bound to the SV40 DNA late in viral infection. Under similar conditions, minichromosomes carrying the rat insulin (rINS1) enhancer, which is under negative regulation in COS-7 cells, bound two proteins which mapped to distinct regions of the rINS1 enhancer. The SV40 P element competed for one of these proteins which bound to the region from -198 to -230. This factor may be related to AP-1. The other factor selectively bound a regulatory element in the region from -92 to -124 of the insulin enhancer. These proteins may play a role in regulating the rINS1 enhancer function.

Animals↗
PubMed1973

Structure of deoxyribonucleic acid on the cell surface during uptake by pneumococcus.

We exposed competent cells of Diplococcus pneumoniae to high-molecular-weight donor deoxyribonucleate (DNA) and examined the state of the DNA bound to them in forms sensitive to deoxyribonuclease I. The portion elutable with 5 M guanidine hydrochloride was shown to be native, of much lower molecular weight (4 x 10(6) to 5 x 10(6)) than the donor, and as active in further transformation as sheared DNA of the same size. The portion resistant to release by guanidine hydrochloride was also shown to be native and active in transformation. These results, along with previous ones, imply that the breaks produced outside the cell are not at genetically specific sites. Furthermore, it was found that entry past the cell barrier to deoxyribonuclease could occur at 0 C by a process sensitive to ethylenediaminetetraacetate.

Carbon Isotopes↗
PubMed1974

Electron microscope and autoradiographic study of ultrastructural aspects of competence and deoxyribonucleic acid absorption in Bacillus subtilis: localization of uptake and of transport of transforming deoxyribonucleic acid in competent cells.

With the aid of serial-section electron microscopy two types of mesosomes can be distinguished in cells of competent cultures of Bacillus subtilis: (i) mesosomes connected to the plasma membrane only (plasma membrane mesosomes) and (ii) mesosomes which extend from the plasma membrane into the nuclear bodies (nuclear mesosomes). Contrary to plasma membrane mesosomes, nuclear mesosomes are absent from the tip zones. Electron microscopic autoradiography of sections of Bacillus subtilis cells exposed to [(3)H]thymidine-labeled transforming deoxyribonucleic acid (DNA) for a short period of time shows that the DNA becomes associated with mesosomes. As a function of time the DNA migrates towards the nucleoids. Transport of DNA is completed within 15 to 60 min after termination of DNA uptake. During its migration the DNA continues to be associated with mesosomes, presumably with nuclear mesosomes. DNA initially associated with plasma membrane mesosomes of the tip zones is probably transported first towards the middle zones peripherally and from there towards the nucleoids.

Absorption↗