Protein phosphorylation is involved in the regulation of chromatin condensation during interphase.

Loci affecting the condensation state of interphase chromatin have been previously identified from analysis of suppression and enhancement of position effect variegation (PEV) in Drosophila. Here we show that Su-var(3)6 and an allelic mutant, e078, which both show suppression of PEV in the heterozygous state, have point mutations (Gly220-->Ser and Gly220-->Asp, respectively) in a protein phosphatase 1 catalytic subunit located at 87B (PP1 87B). The mutated glycine is conserved in all known protein serine/threonine phosphatases in the same gene family, and its substitution decreases PP1 activity. We conclude that protein dephosphorylation by PP1 87B regulates the condensation state of chromatin during interphase.     

Modifiers of position-effect variegation in the region from 86C to 88B of the Drosophila melanogaster third chromosome

Summary Four dominant suppressor and one enhancer of variegation loci were mapped in the polytene chromosome region extending from section 86C to section 88B of the Drosophila melanogaster third chromosome using a set of deficiencies. The suppressor locus Su-var(3) 14 maps in 86CD, Su-var(3) 13 in 86F4-7, Su-var(3)6 in 87B4-7 and Su-var(3)7 in 87E4-5. The enhancer locus E-var(3)3 maps in 87E12-F11. Su-var(3)13, Su-var(3)6 and Su-var(3)7 are also defined by point mutant alleles originally identified by other criteria (Reuter et al. 1986). Duplications covering the suppressor loci Su-var(3)14, Su-var(3)13, Su-var(3)6 and Su-var(3)7 were found to reduce considerably the haplo-abnormal effect of heterozygous point mutants of the corresponding loci. One suppressor locus, Su-var(3)7, maps within a region which has previously been cloned. The positions of deficiency breakpoints delimiting the suppressor locus indicate that all the necessary sequences for its function are located within 10 kb of cloned DNA.     

Butyrate sensitive suppressor of position-effect variegation mutations in drosophila melanogaster

Summary Mutations at a locus on chromosome II of D. melanogaster suppressing position-effect variegation mutations have been identified which display recessive butyrate sensitivity. Survival of homozygous mutant flies is significantly reduced on medium containing sodium n-butyrate. The butyrate sensitive suppressor mutations are further characterized by recessive female sterility and reduced survival of homozygotes. Complementation analysis showed their allelism. The locus of these mutations, Su-var (2) 1, has been localized to 40.5±0.2 and, by using interstitial duplications, to region 31CD on the cytogenetic map. Moreover, the mutant alleles of the Su-var (2) 1 locus display a lethal interaction with the heterochromatic Y chromosome. The presence or absence of a Y chromosome in males or females has a strong influence on the viability of homozygous or transheterozygous suppressor flies. All the genetic properties of Su-var (2) 1 mutants suggest strongly that this locus affects chromosome condensation.     

Protein phosphatase 1 activity in Drosophila mutants with abnormalities in mitosis and chromosome condensation

A Drosophila gene encoding a protein phosphatase 1 (PP1) has been sequenced, and lethal mutations in this locus (87B) analysed. Two mutants (ck19e211 and ck19hs46), which disrupt mitosis, lack the 87B isoenzyme and express only approximately 20% of wild type PP1 activity. The promoter region of the gene is deleted in the ck19e211 mutant. A third mutant (ck19e078), which shows suppression of position effect variegation, but has little effect on mitosis, possesses approximately 35% of wild type PP1 activity. The results indicate that the PP1 87B isoenzyme is involved in regulation of chromosome condensation at interphase as well as mitosis.     

Allelic Variants at the Xanthine Dehydrogenase Locus Affecting Enzyme Activity in DROSOPHILA PSEUDOOBSCURA

Quantitative studies of enzyme activity on gels show about four-fold differences in enzyme activity of different xanthine dehydrogenase (XDH ) alleles. At least three different activity classes could be distinguished among the 23 strains isogenic for the XDH locus. No association of high activity with the high frequency electromorph was observed; instead, the low frequency electromorphs had 0.5 to 2 times the activity of the high frequency electromorph. The frequency of low activity, high activity and intermediate activity XDH alleles among these 23 lines is 0.13, 0.09, and 0.78, respectively.     

Genetic Studies of Mei-1 Gene Activity during the Transition from Meiosis to Mitosis in Caenorhabditis Elegans

Genetic evidence suggests that the mei-1 locus of Caenorhabditis elegans encodes a maternal product required for female meiosis. However, a dominant gain-of-function allele, mei-1(ct46), can support normal meiosis but causes defects in subsequent mitotic spindles. Previously identified intragenic suppressors of ct46 lack functional mei-1 activity; null alleles suppress only in cis but other alleles arise frequently and suppress both in cis and in trans. Using a different screen for suppressors of the dominant ct46 defect, the present study describes another type of intragenic mutation that also arises at high frequency. These latter alleles appear to have reduced meiotic activity and retain a weakened dominant effect. Characterization of these alleles in trans-heterozygous combinations with previously identified mei-1 alleles has enabled us to define more clearly the role of the mei-1 gene product during normal embryogenesis. We propose that a certain level of mei-1 activity is required for meiosis but must be eliminated prior to mitosis. The dominant mutation causes mei-1 activity to function at mitosis; intragenic trans-suppressors act in an antimorphic manner to inactivate multimeric mei-1 complexes. We propose that inactivation of meiosis-specific functions may be an essential precondition of mitosis; failure to eliminate such functions may allow ectopic meiotic activity during mitosis and cause embryonic lethality.     

Mapping of genetic modifiers of Eya1 bor/bor in CAST/EiJ and BALB/cJ that suppress cochlear aplasia and associated deafness

Mice homozygous for the hypomorphic allele Eya1 ( bor ) exhibit cochlear aplasia, with associated deafness, and renal hypoplasia, similar to Branchio-Oto-Renal syndrome (BOR) in humans. Although much is known about the genetics of the disease, little is known about the factors that modify its phenotypic expression. We have recently detailed two modifier loci (Mead1 and Mead2) in a C3HeB/FeJ-Eya1 ( bor/+ ) x C57BL/6 J intercross that suppress the ear-related phenotypes in our hypomorphic mutants. In this study we report corroborating evidence for our initial finding with the identification of two modifier loci mapping to the same region in CAST/EiJ and BALB/cJ. Furthermore, we describe an additional locus (Mead3) on chromosome 19 in CAST/EiJ, within which the previously cloned suppressor Nxf1 resides. The suppression effect on cochlear coiling was studied on congenic line(s) for each protective allele. The penetrance and suppressor strength of these alleles vary by strain and locus. Eya1 ( bor/bor ) hypomorphs, when homozygous for each of the three protective alleles (CAST/EiJ, C57BL/6 J, or BALB/cJ) at the Mead1 or Mead2 locus, exhibit completely penetrant suppression of cochlear agenesis. At the Mead1 locus, the C57BL/6 J and BALB/cJ alleles have comparable strengths. At the Mead2 locus, the C57BL/6 J and CAST/EiJ alleles have comparable strengths. In contrast, mice with genotype Eya1 ( bor/bor )Mead3(CAST/CAST) exhibit incomplete penetrance (50%) and a wide range of cochlear coiling (1/4-1(1/2) turns). The identification of these additional modifier alleles could provide crucial clues for evaluating the candidate genes.     

Molecular and Genetic Organization of the Suppressor of Sable and Minute(1)1b Region in Drosophila Melanogaster

Recessive mutations at the suppressor of sable [su(s)] locus in Drosophila melanogaster result in suppression of second site mutations caused by insertions of the mobile element 412. In order to determine whether su(s) mutations might have other phenotypes, a saturation mapping of the su(s) region was carried out. The screen yielded 76 mutations that comprise ten genetic complementation groups ordered distal to proximal as follows: l(1)1Bh, l(1)1Bi, M(1)1B, su(s), l(1)1Bk, l(1)1Ca, mul, tw, l(1)lDa and brc. Twenty-three of the mutations are su(s) alleles, and all are suppressors of the 412-insertion-caused v1 allele. Although the screen could have detected su(s) mutations causing sex-specific dominant lethality or sterility as well as all types of recessive lethality or sterility, the only other phenotype observed was male sterility that is enhanced by cold temperature. This type of sterility is exhibited only by alleles induced by base-substitution-causing mutagens. Genetic functions of the poly(A+) messages transcribed from the su(s) microregion were identified by the reintroduction of cloned sequences into embryos by P element transformation. su(s) function has been attributed to a 5-kb message. The segment of DNA encoding only this 5-kb message rescues both the suppression and cold-sensitive male sterility phenotypes of su(s). Minute (1) 1B has been provisionally identified as encoding a 3.5-kb message; lethal (1)1Bi encodes a 1-kb message; and lethal (1)1Bk encodes a 4-kb message. The possible functions of su(s) and M(1)1B are discussed.     

Suppressor mutation of position-effect variegation in Drosophila melanogaster affecting chromatin properties

The dominant mutation Su-var(2)1 ⁰¹ which suppresses position-effect variegation and displays recessive butyrate sensitivity was found to result in significant hyperacetylation of histone H4. This biochemical finding, as well as the genetic properties of this mutation, strongly suggest that the wild-type product of the corresponding locus is involved in histone H4 deacetylation. In larvae containing the suppressor mutation the accessibility of chromatin to endogenous nucleases is significantly increased which might be causally connected with histone H4 hyperacetylation. The suppressor mutation Su-var(2)1 ⁰¹ has, therefore, to be classified as a chromatin condensation mutation.     

Drosophila vigilin, DDP1, localises to the cytoplasm and associates to the rough endoplasmic reticulum

Functional characterisation of vigilin, a highly conserved multi-KH-domain protein that binds RNA and ssDNA, remains elusive and, to some extent, controversial. Studies performed in Saccharomyces cerevisiae and human cells indicate that vigilin localises to the cytoplasm, binds ribosomes, associates to RER and regulates mRNA translation. On the other hand, we and others reported a contribution to heterochromatin-mediated gene silencing (PEV) and chromosome segregation in S. cerevisiae, Drosophila and human cells. Whether this contribution is direct remains, however, unclear. Here, we report that Drosophila vigilin, DDP1, vastly localises to the cytoplasm, being largely excluded from the nucleus. We also show that DDP1 preferentially associates to RER and co-purifies with several ribosomal proteins, suggesting a contribution to mRNA translation. In light of these results, the contribution of DDP1 to PEV was re-examined. Here, we show that a newly generated null ddp1(Δ) mutation is only a weak suppressor of PEV, which is in contrast with our own previous results showing dominant suppression in the presence of a strong hypomorphic ddp1(15.1) mutation. Similar results were obtained in the fission yeast Schizosaccharomyces pombe, where vigilin (Vgl1) also associates to RER, having no significant contribution to PEV at centromeres, telomeres and the mating-type locus. Altogether, these results indicate that cytoplasmic localisation and association to RER, but not contribution to heterochromatin organisation, are evolutionarily conserved features of vigilin, favouring a model by which vigilin acts in the cytoplasm, regulating RNA metabolism, and affects nuclear functions only indirectly.     

The Srs2 Suppressor of Rad6 Mutations of Saccharomyces Cerevisiae Acts by Channeling DNA Lesions into the Rad52 DNA Repair Pathway

rad6 mutants of Saccharomyces cerevisiae are defective in the repair of damaged DNA, DNA damage induced mutagenesis, and sporulation. In order to identify genes that can substitute for RAD6 function, we have isolated genomic suppressors of the UV sensitivity of rad6 deletion (rad6 delta) mutations and show that they also suppress the gamma-ray sensitivity but not the UV mutagenesis or sporulation defects of rad6. The suppressors show semidominance for suppression of UV sensitivity and dominance for suppression of gamma-ray sensitivity. The six suppressor mutations we isolated are all alleles of the same locus and are also allelic to a previously described suppressor of the rad6-1 nonsense mutation, SRS2. We show that suppression of rad6 delta is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6 delta SRS2 strain containing an additional mutation in either the RAD51, RAD52, RAD54, RAD55 or RAD57 genes. Possible mechanisms by which SRS2 may channel unrepaired DNA lesions into the RAD52 DNA repair pathway are discussed.     

Loss-of-function alleles of the JIL-1 kinase are strong suppressors of position effect variegation of the wm4 allele in Drosophila

In this article we show that hypomorphic loss-of-function alleles of the JIL-1 histone H3S10 kinase are strong suppressors of position effect variegation (PEV) of the wm4 allele and that lack of JIL-1 activity can counteract the effect of the dominant enhancer Evar2-1 on PEV.     

Genetics of an Unstable White Mutant in Drosophila Simulans: Reversion, Suppression and Somatic Instability

A spontaneous white mutation, white-milky (wmky) of Drosophila simulans is moderately unstable and is associated with a 16-kb long DNA insertion into the white gene. wmky, which is an unstable mutation found in D. simulans, has been genetically analyzed. Among nine spontaneous, partial reversions toward wild type, five were white locus mutations. They are phenotypically different from each other and three show eye color sexual dimorphism indicating a failure of the dosage compensation mechanism. Two w locus mutations whose eye color appeared identical between males and females were also isolated. Of the other back-mutants, three were associated with a recessive suppressor of wmky and one was a semidominant suppressor. These suppressor loci are located on the third chromosome at map positions about 90 and 120, respectively. The suppressor mutations demonstrate specific effects on w locus mutations derived from wmky which lack in the gene dosage compensation. Somatic instability was detected at the frequency of 5.6 X 10(-4) in wmky flies heterozygous for the recessive suppressor and the frequency was increased 10-fold when the suppressor mutation was placed in a different genetic background.     

The tumor suppressor PP2A Abeta regulates the RalA GTPase

The serine-threonine protein phosphatase 2A (PP2A) is a heterotrimeric enzyme family that regulates numerous signaling pathways. Biallelic mutations of the structural PP2A Abeta subunit occur in several types of human tumors; however, the functional consequences of these cancer-associated PP2A Abeta mutations in cell transformation remain undefined. Here we show that suppression of PP2A Abeta expression permits immortalized human cells to achieve a tumorigenic state. Cancer-associated Abeta mutants fail to reverse tumorigenic phenotype induced by PP2A Abeta suppression, indicating that these mutants function as null alleles. Wild-type PP2A Abeta but not cancer-derived Abeta mutants form a complex with the small GTPase RalA. PP2A Abeta-containing complexes dephosphorylate RalA at Ser183 and Ser194, inactivating RalA and abolishing its transforming function. These observations identify PP2A Abeta as a tumor suppressor gene that transforms immortalized human cells by regulating the function of RalA.     

H3S10 phosphorylation by the JIL-1 kinase regulates H3K9 dimethylation and gene expression at the white locus in Drosophila

The JIL-1 kinase is a multidomain protein that localizes specifically to euchromatin interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase. Genetic interaction assays have suggested that the function of the epigenetic histone H3S10ph mark is to antagonize heterochromatization by participating in a dynamic balance between factors promoting repression and activation of gene expression as measured by position-effect variegation (PEV) assays. Interestingly, JIL-1 loss-of-function alleles can act either as an enhancer or indirectly as a suppressor of w(m4) PEV depending on the precise levels of JIL-1 kinase activity. In this study, we have explored the relationship between PEV and the relative levels of the H3S10ph and H3K9me2 marks at the white gene in both wild-type and w(m4) backgrounds by ChIP analysis. Our results indicate that H3K9me2 levels at the white gene directly correlate with its level of expression and that H3K9me2 levels in turn are regulated by H3S10 phosphorylation.     

Suppression of Two Missense Alleles of the TRP5 Locus of SACCHAROMYCES CEREVISIAE

A suppressor SUP101 of alleles trp5-67 and trp5-18 of the trp5 locus of Saccharomyces cerevisiae is described. The two suppressible mutations have been previously classified as missense. The suppression does not result from a physiological bypass of the tryptophan synthetase-catalyzed reaction, since the suppression is allele-specific. IU alleles trp5-70, tryp5-95, and trp5-102; IA alleles trp5-81, trp5-101, and trp5-103; and the ochre alleles trp5-33 and trp5-48 are not suppressed by SUP101. SUP101 does not suppress ochre alleles ade2-1, his5-2, arg4-17, lys1-1, amber alleles trp1-1, tyr7-1, or unclassified alleles at a number of other loci. These results indicate SUP101 is a missense suppressor. Growth on tryptophanless media is dependent upon gene dosage of both the suppressor and the suppressible alleles. Only the diploids homozygous both for the suppressor and suppressible alleles produce growth equivalent to growth of the haploids bearing a suppressible allele and the suppressor. Suppressor-bearing strains grow poorly even on tryptophan-supplemented media. In more than 100 asci analyzed partial growth inhibition on the complete medium always segregated with the suppressor.     

Stf1: Non-Wee Mutations Epistatic to Cdc25 in the Fission Yeast Schizosaccharomyces Pombe

In Schizosaccharomyces pombe, cdc25 is a cell cycle regulated inducer of mitosis. wee1 and phenotypically wee alleles of cdc2 are epistatic to cdc25. Mutant alleles of a new locus, stf1 (suppressor of twenty-five), identified in a reversion analysis of conditionally lethal cdr1-76 cdc25-22 and cdr2-96 cdc25-22 double mutant strains, also suppress both temperature-sensitive and gene disruption alleles of cdc25. These mutants, by themselves, are phenotypically indistinguishable from wild type strains; hence they represent the first known mutations that are epistatic to cdc25 and do not display a wee phenotype. stf1 genetically interacts with other elements of mitotic control in S. pombe. stf1-1 is additive with wee1-50, cdc2-1w and cdc2-3w for suppression of cdc25-22. Also, like wee1- and cdc2-w, stf1- suppression of cdc25 is reversed by overexpression of the putative type 1 protein phosphatase bws1+/dis2+. Interaction with various mutants and plasmid overexpression experiments suggest that stf1 does not operate either upstream or downstream of wee1. Similarly, it does not operate through cdc25 since it rescues the disruption. stf1 appears to encode an important new element of mitotic control.     

Attenuation of ribosomal protein S6 phosphatase activity in chicken embryo fibroblasts transformed by Rous sarcoma virus.

In chicken embryo fibroblasts, phosphorylation of the 40S ribosomal protein S6 increases during G1 but returns to basal level by mitosis. In contrast, in Rous sarcoma virus (RSV)-transformed fibroblasts, S6 remains highly phosphorylated throughout mitosis. This study investigated the mechanism by which RSV alters the pattern of S6 phosphorylation. Pulse-chase experiments demonstrate that phosphate turnover in S6 is rapid in normal cells and in cells infected with an RSV transformation-defective virus. In contrast, phosphate turnover in S6 is severely reduced in cells infected with temperature-sensitive RSV at a temperature permissive for transformation, indicating a diminished S6 phosphatase activity. Fractionation of cell lysates by DEAE chromatography showed an almost threefold lower S6 phosphatase activity in RSV-transformed versus normal cells. The S6 phosphatase was sensitive to inhibitor 2 and specifically recognized by an antibody to type 1 phosphatase (PP1). The S6 phosphatase activity recovered by immunoprecipitation of PP1 was threefold lower in transformed cells, but the steady-state level of expression and the rate of synthesis of PP1 were not altered by oncogenic transformation. Together, the results show that transformation by RSV reduced the S6-PP1 activity.