Allelotyping of follicular thyroid carcinoma: frequent allelic losses in chromosome arms 7q, 11p, and 22q.

The genetic mechanisms involved in development of follicular thyroid carcinoma are poorly understood, although allelic losses (LOH) in this type of tumor have been reported in small panels of follicular thyroid carcinomas examined in earlier studies. To clarify the real frequency of allelic loss we carried out a genome-wide allelotyping study of 66 follicular thyroid carcinomas using 39 microsatellite markers representing all nonacrocentric autosomal arms. The mean frequency of LOH was 9.2%, and the mean fractional allelic loss was 0.09. The most frequent allelic losses were detected in 7q (28%), 11p (28%), and 22q (41%). When we compared these results with our previous allelotyping studies using identical markers in other types of thyroid cancers, the 9.2% mean frequency of allelic loss detected in follicular thyroid carcinomas was higher than that in papillary thyroid carcinomas (3%), but not as high as that detected in anaplastic thyroid carcinomas (20%). Frequent allelic losses of markers on chromosomes 7q, 11p, and 22q suggest locations to examine for the presence of suppressor genes associated with the development of follicular thyroid carcinoma.

Identification of chromosomal deficiencies that modify Drosophila mastermind mutant phenotypes.

Mastermind (Mam) is a component of Notch pathway signaling. In combination with the intracellular domain of Notch and Suppressor of Hairless, Mam forms a transcriptional activation complex. We have initiated a genetic approach to identify other loci involved in Mam function. The screen utilizes engineered mutations in Mam that derive from GAL4-UAS-directed expression of dominant negative constructs. When driven at the wing margin, truncated versions of Mam phenocopy Notch pathway mutations. Correlated with these phenotypes is depression of Notch pathway target expression. Strains expressing truncated versions of Mam were tested for genetic interactions with a large collection of chromosomal deficiencies. Genomic segments that enhanced and suppressed the dominant wing phenotype were identified. These regions may contain uncharacterized loci involved in Notch pathway function.

The Drosophila UBC9 homologue lesswright mediates the disjunction of homologues in meiosis I.

BACKGROUND: In Saccharomyces cerevisiae and other organisms, the UBC9 (ubiquitin-conjugating 9) protein modifies the function of many different target proteins through covalent attachment of the ubiquitin-like protein SMT-3/SUMO. RESULTS: Using a second-site suppression screen of a mutation in the nod locus with a variable meiotic phenotype, we have identified mutations in the Drosophila melanogaster UBC9 homologue, encoded by the gene lesswright (lwr). lwr mutations dominantly suppress the nondisjunction and cytological defects of female meiotic mutations that affect spindle formation. The lwr lethal phenotype is rescued by a Drosophila UBC9/lwr transgene. CONCLUSIONS: We suggest that LWR mediates the dissociation of heterochromatic regions of homologues at the end of meiotic prophase I. Our model proposes that when there is less LWR protein, homologues remain together longer, allowing for more normal spindle formation in mutant backgrounds and therefore more accurate meiotic chromosome segregation.

The dhp1(+) gene, encoding a putative nuclear 5'-->3' exoribonuclease, is required for proper chromosome segregation in fission yeast.

The Schizosaccharomyces pombe dhp1(+) gene is an ortholog of the Saccharomyces cerevisiae RAT1 gene, which encodes a nuclear 5'-->3' exoribonuclease, and is essential for cell viability. To clarify the cellular functions of the nuclear 5'-->3' exoribonuclease, we isolated and characterized a temperature-sensitive mutant of dhp1 (dhp1-1 mutant). The dhp1-1 mutant showed nuclear accumulation of poly(A)(+) RNA at the restrictive temperature, as was already reported for the rat1 mutant. Interestingly, the dhp1-1 mutant exhibited aberrant chromosome segregation at the restrictive temperature. The dhp1-1 cells frequently contained condensed chromosomes, most of whose sister chromatids failed to separate during mitosis despite normal mitotic spindle elongation. Finally, chromosomes were displaced or unequally segregated. As similar mitotic defects were also observed in Dhp1p-depleted cells, we concluded that dhp1(+) is required for proper chromosome segregation as well as for poly(A)(+) RNA metabolism in fission yeast. Furthermore, we isolated a multicopy suppressor of the dhp1-1 mutant, referred to as din1(+). We found that the gene product of dhp1-1 was unstable at high temperatures, but that reduced levels of Dhp1-1p could be suppressed by overexpressing Din1p at the restrictive temperature. Thus, Din1p may physically interact with Dhp1p and stabilize Dhp1p and/or restore its activity.

SPT genes: key players in the regulation of transcription, chromatin structure and other cellular processes.

Suppressor of Ty (SPT) genes were originally identified through a genetic screen for mutations in the yeast Saccharomyces cerevisiae that restore gene expression disrupted by the insertion of the transposon Ty. Classic members of the SPT gene family, SPT11, SPT12, and SPT15, encode for the histones H2A and H2B, and for TATA-binding protein (TBP), respectively. Over the past few years, molecular complexes and cellular functions in which other SPT gene products involve have been discovered through genetic and biochemical studies in yeast and several other organisms: Key regulators of transcription and chromatin structure, such as DSIF, SAGA, and FACT, all contain SPT gene products as essential subunits. In addition, accumulating evidence suggests that SPT gene products play more diverse roles, including roles in DNA replication, DNA recombination and developmental regulation. Here we review the current understanding of the functions and roles of the SPT genes, with special emphasis on the role of SPT5 in transcript elongation and in neuronal development in vertebrates.

[Molecular genetic mechanism of the kidney cancer].

The oncogenic mechanisms of renal cell carcinoma(RCC) are becoming elucidated with recent advances in molecular biology. von Hipple-Lindau disease(VHL) tumor suppressor gene is mutated and inactivated frequently in clear cell type RCCs. The VHL protein forms a complex which shows a ubiquitin ligase activity. The lost of the ubiquitin ligase activity of VHL protein may be a key step for clear cell tumorigenesis. Papillary renal cell carcinomas are caused by activating mutation in the tyrosine kinase domain of the MET gene. This tumorigenic pathway is regulated by c-Src. Immunogene therapies have been started for the patients with advanced RCC. The information based on microarray and Serial Analysis of Gene Expression(SAGE) will provide novel diagnosis and therapy which focus on the tumorigenic mechanism of RCC in the near future.

Hyperthermotolerant fission yeast mutations, sow1 and sow2, suppress the cell cycle defect and stress sensitivity of MAP kinase kinase wis1Delta.

Wis1 is a mitogen-activated protein kinase kinase (MAPKK) that regulates mitosis and mediates stress responses in the fission yeast, Schizosaccharomyces pombe. wis1Delta strains are viable but stress-sensitive and show a mitotic delay. At high temperatures, wis1Delta cells cease division but cellular growth continues. Mutations that suppress the heat sensitivity of a wis1Delta strain were isolated and map to two apparently novel loci, sow1 (for suppressor of wis1Delta) and sow2. In addition to suppressing wis1Delta heat sensitivity, sow1 and sow2 can suppress wis1Delta osmosensitivity and cell cycle defects. sow1 and sow2 mutants in a wis1+ background were able to grow at higher temperatures than wild-type and sow1 showed a mitotic advance. The sow genes may therefore define a novel connection between stress tolerance and cell cycle control.

Gene function analysis by amber stop codon suppression: CMBF is a nuclear protein that supports growth and development of Dictyostelium amoebae.

The C-module-binding factor, CMBF, is a nuclear DNA-binding protein which was originally identified through its specific binding to a promoter element within the retrotransposable element TRE5-A of Dictyostelium discoideum AX2 cells. In order to analyse putative physiological functions of CMBF for the TRE5-A-hosting D. discoideum cells, we used a novel strategy to create mutant cell lines which stably underexpressed functional CMBF. An amber (UAG) translation stop codon was introduced into the chromosomal copy of the CMBF-encoding gene (cbfA), and an amber suppressor tRNA gene was expressed in the same mutant cells. Due to the low efficiency of translation stop codon suppression in this system all recovered cell lines expressed <20 % of wild-type CMBF levels. The mutant cell lines displayed strong growth phenotypes when plated on their natural food source, bacteria. We show evidence that growth reduction was due to impaired phagocytosis of bacteria in the mutants. All obtained mutants showed a strong developmental defect which was defined by the formation of very small fruiting bodies. The strength of the developmental phenotype appeared to depend upon the residual CMBF levels maintained in the mutants. We propose that CMBF is a general transcription regulator which supports the normal expression of several genes required for the maintenance of high proliferation rates of D. discoideum amoebae as well as proper aggregation and development. Our results demonstrate that amber stop codon suppression may be a useful strategy to stably underexpress proteins whose coding genes cannot be successfully disrupted by homologous recombination.

Gen supresor de tumores p53 en neoplasias digestivas / p53 tumor supresor gene in digestive cancer

Malignant diseases of the digestive tract cause more than 50 percent of deaths due to cancer in Chile. There is a high incidence of gastric and gallbladder cancer and an increasing frequency of colorectal cancer. P53 tumor suppressor gene has a great importance in carcinogenesis and its alterations are specially important in digestive tract tumors such as colorectal cancer. There is contradictory evidence about the frequency of p53 gene or protein alterations or their biological significance. There is little information about p53 in Chile and it is mostly limited to immunohistochemical studies. This revision analyzes the frequency of p53 alterations in digestive tract tumors in Chile, using immunohistochemical and molecular biology methods. A special emphasis is given to the prognostic importance of this gene

Pleiotropic resistance to DNA-interactive drugs is associated with increased expression of genes involved in DNA replication, repair, and stress response.

A combination of four genetic suppressor elements (GSEs), two of which are derived from putative transcriptional regulators, was previously found to increase resistance to drugs inhibiting DNA replication in HT1080 fibrosarcoma cells. In the present study, two GSE-transduced cell lines, isolated with and without cytotoxic selection, were found to be resistant to a diverse group of DNA-interactive agents, including aphidicolin, hydroxyurea, cytarabine, etoposide, doxorubicin, and mafosfamide. Changes in gene expression associated with GSE-induced drug resistance were analyzed by cDNA array hybridization and reverse transcription-PCR. Twenty genes were found to be up-regulated in both of the resistant cell lines. These include genes involved in DNA replication and repair (e.g., PCNA, XRCC1, B-MYB, and GADD45), transcriptional regulators associated with stress response, and cell cycle checkpoint control (e.g., YB-1, DBPA, and ATF4), and genes for signal transduction proteins (e.g., protein tyrosine phosphatase 1B and regulatory subunits alpha and beta of cAMP-dependent protein kinase). The observed changes in gene expression may play a role in pleiotropic resistance to different classes of DNA-targeting drugs.

Schizosaccharomyces pombe Rad9 contains a BH3-like region and interacts with the anti-apoptotic protein Bcl-2.

Here we report that the Schizosaccharomyces pombe Rad9 (SpRad9) protein contains a group of amino acids with similarity to the Bcl-2 homology 3 death domain, which is required for SpRad9 interaction with human Bcl-2 and apoptosis induction in human cells. Overexpression of Bcl-2 in S. pombe inhibits cell growth independently of rad9, but enhances resistance of rad9-null cells to methyl methanesulfonate, ultraviolet and ionizing radiation. These observations suggest that SpRad9 may represent the first member of the Bcl-2 protein family identified in yeast, though the cell death pathways in S. pombe may differ from those found in mammals.

Characterization of S818L mutation in HERG C-terminus in LQT2. Modification of activation-deactivation gating properties.

We examined the mechanism(s) for HERG channel dysfunction in an S818L mutation in the HERG C-terminus using the heterologous expression system in Xenopus oocytes. Injection of S818L cRNA alone did not produce expressed currents. Coinjection of an equal amount of S818L cRNA with wild-type (WT) cRNA into oocytes did not exhibit apparent dominant-negative suppression. However, coinjection of excess amounts of S818L cRNAs with WT cRNA into oocytes decreased HERG current amplitudes and shifted the voltage dependence of activation to negative potentials, accelerated its activation and deactivation. The data suggest that S818L alone cannot form functional channels, whereas S818L subunits can, at least in part, coassemble with WT subunits to form heterotetrameric functional channels, and imply that the HERG C-terminus may contain a domain involving the activation-deactivation process of the channel. These findings may provide new insights into the structure-function relationships of the HERG C-terminus.

Interactions between activating region 3 of the Escherichia coli cyclic AMP receptor protein and region 4 of the RNA polymerase sigma(70) subunit: application of suppression genetics.

The Escherichia coli cyclic AMP receptor protein, CRP, induces transcription at Class II CRP-dependent promoters by making three different activatory contacts with different surfaces of holo RNA polymerase. One contact surface of CRP, known as Activating Region 3 (AR3), is functional in the downstream subunit of the CRP dimer and is predicted to interact with region 4 of the RNAP sigma(70) subunit. We have previously shown that a mutant CRP derivative that activates transcription primarily via AR3, CRP HL159 KE101 KN52, requires the positively charged residues K593, K597 and R599 in sigma(70) for activation. Here, we have used the positive control substitution, EK58, to disrupt AR3-dependent activation by CRP HL159 KE101 KN52. We then screened random mutant libraries and an alanine scan library of sigma(70) for candidates that restore activation by CRP HL159 KE101 KN52 EK58. We found that changes at R596 and R599 in sigma(70) can restore activation by CRP HL159 KE101 KN52 EK58. This suggests that the side-chains of both R596 and R599 in sigma(70) clash with K58 in CRP. Maximal activation by CRP HL159 KE101 KN52 EK58 is achieved with the substitutions RE596 or RD596 in sigma(70). We propose that there are specific charge-charge interactions between E596 or D596 in sigma(70) and K58 in AR3. Thus, no increase in activation is observed in the presence of another positive control substitution, EG58 (CRP HL159 KE101 KN52 EG58). Similarly, both sigma(70) RE596 and sigma(70) RD596 can restore activation by CRP EK58 but not CRP EG58, and they both decrease activation by wild-type CRP. We suggest that E596 and D596 in sigma(70) can positively interact with K58 in AR3, thereby enhancing activation, but negatively interact with E58, thereby decreasing activation. The substitution, KA52 in AR3 increases Class II CRP-dependent activation by removing an inhibitory lysine residue. However, this increase is not observed in the presence of either sigma(70) RE596 or sigma(70) RD596. We conclude that the inhibitory side-chain, K52 in AR3, clashes with R596 in sigma(70). Finally, we show that the sigma(70) RE596 and RD596 substitutions affect CRP-dependent activation from Class II, but not Class I, promoters.

Expression of "suppressor of cytokine signalling" (SOCS) genes in the developing and adult mouse nervous system.

Growth factor and cytokine signalling in the developing nervous system has multiple effects, ranging from cell differentiation and cell survival to modulation of cell phenotype. Molecules that can regulate growth factor signalling pathways will therefore be of importance in determining the cellular response to factor stimulation. Members of a recently described gene family, the suppressor of cytokine signalling (SOCS) family, can regulate signalling events downstream of predominantly cytokine stimulation and may have important roles in the nervous system. We have examined the temporal and spatial expression of SOCS-1, SOCS-2, and SOCS-3 in the developing and adult nervous system by use of Northern analysis and in situ hybridisation. All three genes were expressed in the brain, with maximal expression from embryonic day 14 to postnatal day 8 and declining thereafter, with SOCS-2 being the most highly expressed. In situ hybridisation analysis showed that SOCS-1 and SOCS-3 had a low and widespread pattern of expression, whereas SOCS-2 expression was higher and tightly regulated. Its expression pattern indicated that SOCS-2 was expressed exclusively in neurons and that it was switched on developmentally at the time of neuronal differentiation.

tRNA leucine identity and recognition sets.

Transfer RNAs (tRNAs) are grouped into two classes based on the structure of their variable loop. In Escherichia coli, tRNAs from three isoaccepting groups are classified as type II. Leucine tRNAs comprise one such group. We used both in vivo and in vitro approaches to determine the nucleotides that are required for tRNA(Leu) function. In addition, to investigate the role of the tRNA fold, we compared the in vivo and in vitro characteristics of type I tRNA(Leu) variants with their type II counterparts.A minimum of six conserved tRNA(Leu) nucleotides were required to change the amino acid identity and recognition of a type II tRNA(Ser) amber suppressor from a serine to a leucine residue. Five of these nucleotides affect tRNA tertiary structure; the G15-C48 tertiary "Levitt base-pair" in tRNA(Ser) was changed to A15-U48; the number of nucleotides in the alpha and beta regions of the D-loop was changed to achieve the positioning of G18 and G19 that is found in all tRNA(Leu); a base was inserted at position 47n between the base-paired extra stem and the T-stem; in addition the G73 "discriminator" base of tRNA(Ser) was changed to A73. This minimally altered tRNA(Ser) exclusively inserted leucine residues and was an excellent in vitro substrate for LeuRS. In a parallel experiment, nucleotide substitutions were made in a glutamine-inserting type I tRNA (RNA(SerDelta); an amber suppressor in which the tRNA(Ser) type II extra-stem-loop is replaced by a consensus type I loop). This "type I" swap experiment was successful both in vivo and in vitro but required more nucleotide substitutions than did the type II swap. The type I and II swaps revealed differences in the contributions of the tRNA(Leu) acceptor stem base-pairs to tRNA(Leu) function: in the type I, but not the type II fold, leucine specificity was contingent on the presence of the tRNA(Leu) acceptor stem sequence. The type I and II tRNAs used in this study differed only in the sequence and structure of the variable loop. By altering this loop, and thereby possibly introducing subtle changes into the overall tRNA fold, it became possible to detect otherwise cryptic contributions of the acceptor stem sequence to recognition by LeuRS. Possible reasons for this effect are discussed.

The zebrafish bonnie and clyde gene encodes a Mix family homeodomain protein that regulates the generation of endodermal precursors.

Vertebrate endoderm development has recently become the focus of intense investigation. In this report, we first show that the zebrafish bonnie and clyde (bon) gene plays a critical early role in endoderm formation. bon mutants exhibit a profound reduction in the number of sox17-expressing endodermal precursors formed during gastrulation, and, consequently, a profound reduction in gut tissue at later stages. The endodermal precursors that do form in bon mutants, however, appear to differentiate normally indicating that bon is not required at later steps of endoderm development. We further demonstrate that bon encodes a paired-class homeodomain protein of the Mix family that is expressed transiently before and during early gastrulation in both mesodermal and endodermal progenitors. Overexpression of bon can rescue endodermal gene expression and the formation of a gut tube in bon mutants. Analysis of a newly identified mutant allele reveals that a single amino acid substitution in the DNA recognition helix of the homeodomain creates a dominant interfering form of Bon when overexpressed. We also show through loss- and gain-of-function analyses that Bon functions exclusively downstream of cyclops and squint signaling. Together, our data demonstrate that Bon is a critical transcriptional regulator of early endoderm formation.

Impact of human immunodeficiency virus type 1 RNA dimerization on viral infectivity and of stem-loop B on RNA dimerization and reverse transcription and dissociation of dimerization from packaging.

The kissing-loop domain (KLD) encompasses a stem-loop, named kissing-loop or dimerization initiation site (DIS) hairpin (nucleotides [nt] 248 to 270 in the human immunodeficiency virus type 1 strains HIV-1(Lai) and HIV-1(Hxb2)), seated on top of a 12-nt stem-internal loop called stem-loop B (nt 243 to 247 and 271 to 277). Destroying stem-loop B reduced genome dimerization by approximately 50% and proviral DNA synthesis by approximately 85% and left unchanged the dissociation temperature of dimeric genomic RNA. The most affected step of reverse transcription was plus-strand DNA transfer, which was reduced by approximately 80%. Deleting nt 241 to 256 or 200 to 256 did not reduce genome dimerization significantly more than the destruction of stem-loop B or the DIS hairpin. We conclude that the KLD is nonmodular: mutations in stem-loop B and in the DIS hairpin have similar effects on genome dimerization, reverse transcription, and encapsidation and are also "nonadditive"; i.e., a larger deletion spanning both of these structures has the same effects on genome dimerization and encapsidation as if stem-loop B strongly impacted DIS hairpin function and vice versa. A C258G transversion in the palindrome of the kissing-loop reduced genome dimerization by approximately 50% and viral infectivity by approximately 1.4 log. Two mutations, CGCG261-->UUAA261 (creating a weaker palindrome) and a Delta241-256 suppressor mutation, were each able to reduce genome dimerization but leave genome packaging unaffected.

Respective roles of cyclobutane pyrimidine dimers, (6-4)photoproducts, and minor photoproducts in ultraviolet mutagenesis of repair-deficient xeroderma pigmentosum A cells.

The role of UV light-induced photoproducts in initiating base substitution mutation in human cells was examined by determining the frequency and spectrum of mutation in a supF tRNA gene in a shuttle vector plasmid transfected into DNA repair deficient cells (xeroderma pigmentosum complementation group A). To compare the role of two major UV-induced photoproducts, cis-syn cyclobutane-type pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), each photoproduct was removed from UV-irradiated plasmid by photoreactivation before transfection. Removal of either CPDs or 6-4PPs by in vitro photoreactivation reduced the mutation frequency while keeping the mutation distribution and the predominance of G:C-A:T transitions as UV-irradiated plasmid without photoreactivation, indicating that both cytosine-containing CPDs and 6-4PPs were premutagenic lesions for G:C-A:T transitions. On the other hand, A:T-G:C transitions were not recovered from plasmids after the removal of 6-4PPs, whereas this type of mutation occurred at a significant level (11%) after the removal of CPDs. Thus, the premutagenic lesions for the A:T-G:C transition are 6-4PPs. Removal of both CPDs and 6-4PPs resulted in the disappearance of mutational hot spots and random distribution of mutation as observed in unirradiated control plasmids. However, the mutational spectrum of photoreactivated plasmids differed significantly from that of unirradiated plasmids. A characteristic feature is a high portion of A:T-T:A transversions (11%) in the photoreactivated plasmid. This mutation is due to nondipyrimidinic "minor" photoproducts, and the mutation spectrum suggests that TA*, the major photoproduct of thymidylyl-(3'-5')-deoxyadenosine, is the premutagenic lesion for this mutation. This is the first report revealing the distinct mutagenic roles of the major UV photoproducts and "minor" photoproducts by the use of (6-4)photolyase.

Intragenic and intergenic suppression of the Escherichia coli ATP synthase subunit a mutation of Gly-213 to Asn: functional interactions between residues in the proton transport site.

Subunit a of the ATP synthase F(o) sector contains a transmembrane helix that interacts with subunit c and is critical for H(+) transport activity. From a cysteine scan in the region around the essential subunit a residue, Arg-210, we found that the replacement of aGly-213 greatly attenuated ATP hydrolysis, ATP-dependent proton pumping and Delta mu(H)+-dependent ATP synthesis. Various amino acid substitutions caused similar effects, suggesting that functional perturbations were caused by altering the environment or conformation of aArg-210. aG213N, which was particularly severe in effect, was suppressed by two second-site mutations, aL251V and cD61E. These mutations restored efficient coupling; the latter also increased ATP-dependent proton transport rates. These results were consistent with the proposed functional interaction between aArg-210 and cAsp-61, the likely carrier of the transported proton. From Arrhenius analysis of steady-state ATP hydrolytic activity, the transport mutants had large increases in the transition-state enthalpic and entropic parameters. Linear isokinetic relationships demonstrate that the transport mechanism is coupled to the rate-limiting catalytic transition-state step, which we have previously shown to involve the rotation of the gamma subunit in multi-site, co-operative catalysis.