Rapid development of post-radiotherapy sarcoma and breast cancer in a patient with a novel germline 'de-novo' TP53 mutation.

AIMS: Germline mutations in the TP53 tumour suppressor gene are associated with Li-Fraumeni syndrome, which is characterised by a spectrum of neoplasms occurring in children and young adults that predominantly include early-onset breast cancer, a variety of sarcomas, brain tumours and adrenocortical tumours. The identification of patients carrying TP53 mutations is primarily based on a positive family history of these early-onset characteristic cancer types. The aim of this study is to emphasize the importance of TP53 molecular testing in patients with very early onset breast cancer and no family history of cancer. MATERIALS AND METHODS: A young woman with no family history of cancer presented with bilateral breast cancer at the age of 27 years. Forty months later she developed malignant fibrous histiocytoma of the right clavicle and another primary left breast cancer. Molecular testing of mutations 185delAG, 5382insC in BRCA1 gene and 6174delT in BRCA2 gene was performed using multiplex PCR and separation on a denaturing polyacrylamide gel. TP53 molecular analysis was performed by PCR-SSCP analysis of the whole coding region of the TP53. Exon 8 PCR products were sequenced using an ABI dye terminator kit and examined on an ABI 3100 automated sequencer. RESULTS: Molecular testing of peripheral blood DNA did not reveal mutations in BRCA1 or BRCA2 genes. A novel germline TP53 mutation, c.G841C, p.D281N, was identified. The detected mutation is a missense substitution, c.G841C, resulting in the substitution of the amino acid aspartate to asparagine, p.D281N. Molecular analysis in her parents showed that neither of them carried the mutation. CONCLUSIONS: We describe a novel 'de novo'TP53 mutation and discuss the importance of molecular testing in early-onset breast cancer patients and its effect on the management and outcome of the disease.

Elevated expression of the CD4 receptor and cell cycle arrest are induced in Jurkat cells by treatment with the novel cyclic dinucleotide 3',5'-cyclic diguanylic acid.

The effect of the novel, naturally occurring nucleotide cyclic diguanylic acid (c-di-GMP) on the lymphoblastoid CD4+ Jurkat cell line was studied. When exposed to 50 microM c-di-GMP, Jurkat cells exhibited a markedly elevated expression of the CD4 receptor of up to 6.3-fold over controls. C-di-GMP also causes blockage of the cell cycle at the S-phase, characterized by increased cellular thymidine uptake, reduction in G2/M-phase cells, increase in S-phase cells and decreased cell division. Additionally c-di-GMP naturally enters these cells and binds irreversibly to the P21ras protein. The effects described appear to be unique for c-di-GMP.

c-di-GMP-binding protein, a new factor regulating cellulose synthesis in Acetobacter xylinum.

A protein which specifically binds cyclic diguanylic acid (c-di-GMP), the reversible allosteric activator of the membrane-bound cellulose synthase system of Acetobacter xylinum, has been identified in membrane preparations of this organism. c-di-GMP binding is of high affinity (KD 20 nM), saturable and reversible. The equilibrium of the reaction is markedly and specifically shifted towards the binding direction by K+. The c-di-GMP binding protein, structurally associated with the cellulose synthase, appears to play a major role in modulating the intracellular concentration of free c-di-GMP and thus may constitute an essential factor in regulating cellulose synthesis in vivo.

Genetic data indicate that proteins containing the GGDEF domain possess diguanylate cyclase activity.

A conserved domain, called GGDEF (referring to a conserved central sequence pattern), is detected in many procaryotic proteins, often in various combinations with putative sensory-regulatory components. Most sequenced bacterial genomes contain several different GGDEF proteins. The function of this domain has so far not been experimentally shown. Through genetic complementation using genes from three different bacteria encoding proteins with GGDEF domains as the only element in common, we present genetic data indicating (a) that the GGDEF domain is responsible for the diguanylate cyclase activity of these proteins, and (b) that the activity of cellulose synthase in Rhizobium leguminosarum bv. trifolii and Agrobacterium tumefaciens is regulated by cyclic di-GMP as in Acetobacter xylinum.

Cyclic diguanylic acid and cellulose synthesis in Agrobacterium tumefaciens.

The occurrence of the novel regulatory nucleotide bis(3',5')-cyclic diguanylic acid (c-di-GMP) and its relation to cellulose biogenesis in the plant pathogen Agrobacterium tumefaciens was studied. c-di-GMP was detected in acid extracts of 32P-labeled cells grown in various media, and an enzyme responsible for its formation from GTP was found to be present in cell-free preparations. Cellulose synthesis in vivo was quantitatively assessed with [14C]glucose as a tracer. The organism produced cellulose during growth in the absence of plant cells, and this capacity was retained in resting cells. Synthesis of a cellulosic product from UDP-glucose in vitro with membrane preparations was markedly stimulated by c-di-GMP and its precursor GTP and was further enhanced by Ca2+. The calcium effect was attributed to inhibition of a c-di-GMP-degrading enzyme shown to be present in the cellulose synthase-containing membranes.

Characterization of Vibrio fischeri rRNA operons and subcloning of a ribosomal DNA promoter.

Analysis of rRNA genes in Vibrio fischeri indicates the presence of eight rRNA gene sets in this organism. It was found that the genes for 5S rRNA, 16S rRNA, and 23S rRNA are organized in operons in the following order: 5' end 16S rRNA 23S RNA 5S rRNA 3' end. Although the operons are homologous, they are not identical with regard to cleavage sites for various restriction endonucleases. A DNA library was constructed, and three ribosomal DNA clones were obtained. One of these clones contained an entire rRNA operon and was used as a source for subcloning. The promoter region which leads to plasmid instability was successfully subcloned into pHG165. The terminator region was subcloned into pBR322.

Ribosomal RNA genes in Mycoplasma.

Using Southern blotting analysis with labelled mycoplasmal ribosomal RNA as probe, two fragments (1 Kb and 5 Kb) were detected in an EcoR I digest of Mycoplasma capricolum DNA. This analysis revealed that the 5 Kb fragment carries both 16S rRNA sequences and the entire 23S rRNA gene of this mycoplasma. The 1 Kb fragment contains 16S rRNA sequences only. The 5 Kb EcoR I fragment has been cloned and used to characterize the structure of rRNA cistrons in various Mycoplasma strains. These experiments clearly demonstrate a substantial homology of Mycoplasma capricolum rRNA sequences with the E. coli rRNA cistron on one hand, and with Mycoplasma mycoides subsp. capri and Acholeplasma laidlawii on the other hand. This analysis also reveals two rRNA cistrons in Mycoplasma mycoides subsp. capri and Acholeplasma laidlawii whereas one rRNA cistron is present in Mycoplasma capricolum.

Molecular and biological features of mollicutes (mycoplasmas).

The small size of the mollicute genome considerably restricts the amount of genetic information available to the organisms. This is reflected in the relatively small number of cell proteins synthesized, the lack of many biosynthetic pathways and the marked dependence on exogenous nutrients for growth. The protein synthesizing machinery of mollicutes resembles that of eubacteria and is sensitive to the same antibiotics, except for rifampicin, to which RNA polymerases of mollicutes appear resistant. The mollicute ribosomes are built of 50 S and 30 S subunits and contain about 50 different proteins and 5 S, 16 S and 23 S rRNA, as in eubacteria. However, the 5 S rRNA in mollicutes appears shorter (107-112 nucleotides) than in eubacteria (116-120 nucleotides). We hybridized restriction endonuclease-digested DNA from a variety of Mycoplasma, Ureaplasma, Acholeplasma and Spiroplasma species with nick-translated probes consisting of defined portions of the rrnB rRNA operon of Escherichia coli and the rRNA operon of M. capricolum. The results suggest the presence of only one or two sets (operons) of rRNA genes in the genome of Mollicutes, a number falling considerably below that of the eubacteria examined so far but resembling that found in archaebacteria. Our data also indicate a marked nucleotide sequence homology along the rrnB rRNA operon of E. coli and the rRNA operons of the various mollicutes, indicating that the rRNA genes in mollicutes are linked in the classical prokaryotic fashion 16 S-23 S-5 S. Each mollicute appeared to possess, on its genome, different flanking sequences adjacent to the rRNA operon(s), resulting in species-specific hybridization patterns.(ABSTRACT TRUNCATED AT 250 WORDS)

Mycoplasmas (Mollicutes) have a low number of rRNA genes.

DNA from Mycoplasma, Ureaplasma, Acholeplasma, and Spiroplasma species digested by restriction endonucleases was hybridized with probes consisting of portions of the rrnB rRNA operon of Escherichia coli and the rRNA operon of Mycoplasma capricolum. The results indicate the presence of only one or two sets of rRNA genes in the genome of Mollicutes linked in the procaryotic fashion, 16S-23S-5S.

Physical mapping of the ribosomal RNA genes of Mycoplasma capricolum.

Physical mapping of the rRNA genes of Mycoplasma capricolum was done by digestion of the mycoplasmal DNA with EcoRI, PstI and BglII and hybridization with nick-translated probes consisting of defined portions of the rrnB ribosomal RNA operon of Escherichia coli. The results indicate that the rRNA genes in the chromosome of M. capricolum are arranged in two clusters, each organized in the order 5'-16S-23S-5S-3', resembling the order of the genes in the rrnB operon, with no large spacer regions separating the genes in each cluster.

Mycoplasmal ribosomal RNA genes and their use as probes for detection and identification of Mollicutes.

The number and organization of ribosomal RNA (rRNA) genes in the genome of Mycoplasma, Ureaplasma, Acholeplasma and Spiroplasma species were studied by the Southern hybridization technique. Restriction endonuclease-digested DNAs of the organisms were hybridized with nick-translated probes consisting of defined portions of the rrnB rRNA operon of Escherichia coli and with a recombinant plasmid pMC5 constructed of pBR325 and an insert containing M. capricolum genes for 23S, 5S and most of the 16S rRNA gene. The hybridization data indicate the presence of only one or two sets of rRNA genes in the mollicutes tested, a number lower than in eubacteria. The rRNA genes in mollicutes appear to be organized as clusters (acting apparently as operons) in the typical prokaryotic fashion, 5'-16S-23S-5S-3'. Despite the marked sequence homology shared by the rRNA operons of the different mollicutes and of E. coli, the operons are not identical. Thus, there is an EcoRI restriction site in the 16S rRNA genes in only 8 of the 13 species tested. The recombinant plasmid pMC5 has provided a sensitive probe for detection and identification of mollicutes in contaminated cell cultures. The purified DNA of the tested cell culture, or its supernatant fluid, was digested by EcoRI, and Southern blot hybridization of the products was performed with nick-translated pMC5. The probe did not hybridize with eukaryotic DNA. Each of the mollicutes species examinated exhibited a species-specific hybridization pattern. The hybridization tests enabled the identification of the four most prevalent mycoplasma contaminants of cell cultures, M. orale, M. hyorhinis, M. arginini and A. laidlawii. The test is capable of detecting 1 ng of mycoplasmal DNA, roughly equivalent to the DNA content of 10(5) mycoplasmas. The possibility of using this approach for detection and identification of noncultivable mycoplasmas in plant and insect tissues is under investigation.

The novel cyclic dinucleotide 3'-5' cyclic diguanylic acid binds to p21ras and enhances DNA synthesis but not cell replication in the Molt 4 cell line.

1. The effect of the novel, naturally occurring nucleotide 3'-5' cyclic diguanylic acid (c-di-GMP) on the lymphoblastoid Molt 4 cell line was studied. When exposed to this guanine nucleotide. Molt 4 cells exhibited a marked increase in [3H]thymidine incorporation, up to 200-fold at 50 microM c-di-GMP. Correspondingly, the DNA content of the treated cells was 9-fold higher than untreated cells. Stimulation of [3H]thymidine incorporation into the cells was time- and concentration-dependent. This effect was specific and was not observed with GMP or cyclic GMP, nor with the unhydrolysable GTP analogues, guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta gamma-imido]-triphosphate. C-di-GMP entrance into the cells was experimentally verified and occurred without using any means of cell permeabilization. SDS/PAGE analysis of cells exposed to [32P]c-di-GMP, followed by autoradiography, revealed the labelling of three low-molecular-mass proteins at 18-27 kDa. The labelling is highly specific to c-di-GMP and its extent was not affected by other guanine nucleotides. 2. One of the c-di-GMP-binding proteins was found to be the p21ras protein, by immunoprecipitation with the anti-Ras monoclonal antibody Y13-259. The effects described appear to be unique for c-di-GMP and, taken together, raise the possibility that an irreversible binding of this guanine nucleotide to the growth-promoting p21ras protein results in a fixed active conformation of this protein affecting DNA synthesis. Strikingly, although at 48 h of growth markedly high DNA levels were found in Molt 4 cells treated with c-di-GMP, this guanine nucleotide had no effect on cell replication during this period. Thus Molt 4 cells exposed to c-di-GMP enter the S phase uncoordinated with their overall replication rate.

A correlation between the expression of the bcr-abl chimeric gene and severity of the clinical state of CML patients with time.

Using the reverse polymerase chain reaction, four Ph' positive CML patients were followed for 2 years; a correlation between the severity of the clinical state and the b3a2 expression was noted with time. Additionally, amplification of the c-myc proto-oncogene was observed, using Southern blot analysis, in one patient prior to his entry to the blast phase. No reorganization of the bcr-abl rearrangement site was found in the latter patient. The data suggest that a routine follow-up of CML patients using the Southern blot analysis and the reverse transcription-polymerase chain reaction might be of importance in evaluating the progression of the disease.

The use of DNA markers in the pre-clinical diagnosis of familial adenomatous polyposis.

Familial adenomatous polyposis (FAP), an autosomal dominant inherited disease, confers a high risk of colon cancer. For presymptomatic diagnosis of FAP, we performed linkage studies in three unrelated Israeli families with FAP, using seven polymorphic systems around or at the APC locus on chromosome 5q. These systems are constituted of three DNA probes, recognizing four restriction fragment length polymorphism: C11p11, YN5.48 and pi227; three cytosine-adenine repeat markers: D5S318, D5S346 and MBC; and one intragenic polymorphism: APC-SspI. A meiotic recombination event was detected, apparently between the FAP gene and probe pi227. Based on the different analysis systems, we determined the haplotype at the APC locus in 11 at-risk individuals of the three families, six of whom were found to carry the disease-linked allele. Additionally, we identified a new FAP patient, in whom sigmoidoscopy showed the presence of adenomatous polyps throughout the colon.

Molecular analysis of an asymptomatic Ph-positive CML patient with 27 years of prolonged remission.

We describe the results of clinical, cytogenetic, and molecular biological studies of a patient who, after two courses of treatment with Busulphan, has remained free of symptoms of Philadelphia chromosome-positive chronic myeloid leukemia for 27 years, except for the persistence of a Ph1 chromosome and the presence of its transcribed chimeric mRNA.

Characterization of the mycoplasma genome.

Recent advances on the properties of the mycoplasma genome, including size, base composition, replication, extrachromosomal DNA, and transfer of genetic material are briefly reviewed, with emphasis on their phylogenetic implications. The use of cleavage patterns of the mycoplasma genome by restriction endonucleases as "finger-prints" indicating genetic relatedness among strains is discussed. The data support the notion that strains of mycoplasma species of strict host and tissue specificity exhibit marked genetic homogeneity, suggesting a clonal origin for some species. The regions of the mycoplasma genome carrying the ribosomal RNA (rRNA) genes have been studied using restriction endonucleases, cloning, and hybridization procedures. The mycoplasmal rRNA cistrons cross-hybridized among themselves, and with the seven rRNA cistrons of Escherichia coli, demonstrating the marked conservation of structure during evolution of this part of the procaryotic genome. In most of the mollicutes tested so far the number of rRNA cistrons is two, but a few species appear to carry only one rRNA cistron in their genome.

Cloning of a gene involved in cellulose biosynthesis in Acetobacter xylinum: complementation of cellulose-negative mutants by the UDPG pyrophosphorylase structural gene.

Three cellulose-negative (Cel-) mutants of Acetobacter xylinum strain ATCC 23768 were complemented by a cloned 2.8 kb DNA fragment from the wild type. Biochemical analysis of the mutants showed that they were deficient in the enzyme uridine 5'-diphosphoglucose (UDPG) pyrophosphorylase. The analysis also showed that the mutants could synthesize beta(1-4)-glucan in vitro from UDPG, but not in vivo from glucose. This result was expected, since UDPG is known to be the precursor for cellulose synthesis in A. xylinum. In order to analyze the function of the cloned gene in more detail, its biological activity in Escherichia coli was studied. These experiments showed that the cloned fragment could be used to complement an E. coli mutant deficient in the structural gene for UDPG pyrophosphorylase. It is therefore clear that the cloned fragment must contain this gene from A. xylinum. This is to our knowledge the first example of the cloning of a gene with a known function in cellulose biosynthesis from any organism, and we suggest the gene be designated celA.

Phosphodiesterase A1, a regulator of cellulose synthesis in Acetobacter xylinum, is a heme-based sensor.

The phosphodiesterase A1 protein of Acetobacter xylinum, AxPDEA1, is a key regulator of bacterial cellulose synthesis. This phosphodiesterase linearizes cyclic bis(3'-->5')diguanylic acid, an allosteric activator of the bacterial cellulose synthase, to the ineffectual pGpG. Here we show that AxPDEA1 contains heme and is regulated by reversible binding of O(2) to the heme. Apo-AxPDEA1 has less than 2% of the phosphodiesterase activity of holo-AxPDEA1, and reconstitution with hemin restores full activity. O(2) regulation is due to deoxyheme being a better activator than oxyheme. AxPDEA1 is homologous to the Escherichia coli direct oxygen sensor protein, EcDos, over its entire length and is homologous to the FixL histidine kinases over only a heme-binding PAS domain. The properties of the heme-binding domain of AxPDEA1 are significantly different from those of other O(2)-responsive heme-based sensors. The rate of AxPDEA1 autoxidation (half-life > 12 h) is the slowest observed so far for this type of heme protein fold. The O(2) affinity of AxPDEA1 (K(d) approximately 10 microM) is comparable to that of EcDos, but the rate constants for O(2) association (k(on) = 6.6 microM(-)(1) s(-)(1)) and dissociation (k(off) = 77 s(-)(1)) are 2000 times higher. Our results illustrate the versatility of signal transduction mechanisms for the heme-PAS class of O(2) sensors and provide the first example of O(2) regulation of a second messenger.

The cyclic diguanylic acid regulatory system of cellulose synthesis in Acetobacter xylinum. Chemical synthesis and biological activity of cyclic nucleotide dimer, trimer, and phosphothioate derivatives.

An unusual compound, cyclic-bis(3'----5') diguanylic acid (c-di-GMP or cGpGp), is involved in the regulation of cellulose synthesis in the bacterium Acetobacter xylinum. This cyclic dinucleotide acts as an allosteric, positive effector of cellulose synthase activity in vitro (Ka = 0.31 microM) and is inactivated via degradation by a Ca2(+)-sensitive phosphodiesterase, PDE-A (Km = 0.25 microM). A series of 13 analogs cyclic dimer and trimer nucleotides were synthesized, employing a phosphotriester approach, and tested for the ability to mimick c-di-GMP as activators of cellulose synthase and as substrates for PDE-A. Seven of the synthetic compounds stimulate cellulose synthase activity and all of these activators undergo the Ca2(+)-inhibited degradation reaction. The order of affinities for synthase activators is cGpGp approximately cdGpGp approximately cGp(S)Gp (S-diastereomer) greater than cIpGp greater than cdGpdGp greater than cXpGp greater than cIpIp greater than cGp(S)Gp (R-diastereomer). Three cyclic dinucleotides of negligible affinity for either enzyme are cApAp, cUpUp, and cCpCp. This same order of affinities essentially pertains to the analogs as inhibitors of PDE-A activity, but at least one cyclic dinucleotide, cXpXp, which does not bind to cellulose synthase, is also a substrate for the degradation reaction, demonstrating that although the two enzymes share a similar, high degree of specificity for c-di-GMP, their cyclic dinucleotide binding sites are not identical. Phosphodiester bonds of activators in which an exocyclic oxygen is replaced with an atom of sulfur (cGp(S)Gp isomers) resist the action of PDE-A, and such derivatives may be prototypes for synthetic non-hydrolyzable c-di-GMP analogs.

Polypeptide composition of bacterial cyclic diguanylic acid-dependent cellulose synthase and the occurrence of immunologically crossreacting proteins in higher plants.

To comprehend the catalytic and regulatory mechanism of the cyclic diguanylic acid (c-di-GMP)-dependent cellulose synthase of Acetobacter xylinum and its relatedness to similar enzymes in other organisms, the structure of this enzyme was analyzed at the polypeptide level. The enzyme, purified 350-fold by enzyme-product entrapment, contains three major peptides (90, 67, and 54 kDa), which, based on direct photoaffinity and immunochemical labeling and amino acid sequence analysis, are constituents of the native cellulose synthase. Labeling of purified synthase with either [32P]c-di-GMP or [alpha-32P]UDP-glucose indicates that activator- and substrate-specific binding sites are most closely associated with the 67- and 54-kDa peptides, respectively, whereas marginal photolabeling is detected in the 90-kDa peptide. However, antibodies raised against a protein derived from the cellulose synthase structural gene (bcsB) specifically label all three peptides. Further, the N-terminal amino acid sequences determined for the 90- and 67-kDa peptides share a high degree of homology with the amino acid sequence deduced from the gene. We suggest that the structurally related 67- and 54-kDa peptides are fragments proteolytically derived from the 90-kDa peptide encoded by bcsB. The anti-cellulose synthase antibodies crossreact with a similar set of peptides derived from other cellulose-producing microorganisms and plants such as Agrobacterium tumefaciens, Rhizobium leguminosarum, mung bean, peas, barley, and cotton. The occurrence of such cellulose synthase-like structures in plant species suggests that a common enzymatic mechanism for cellulose biogenesis is employed throughout nature.