Circadian clock phenotypes of chromosome aberrations with a breakpoint at the per locus

Summary The circadian rhythm phenotypes of eight chromosome aberrations with a breakpoint in the region of the per locus (3B1-2) were analyzed. Two duplications and five deficiencies with a 3B1-2 breakpoint produce either a wild-type or an arrhythmic clock phenotype while one translocation with a 3B1-2 breakpoint, T(1;4)JC43, produces locomotor-activity rhythms with either very-long period (31–39 h), rhythms that grade into arrhythmicity, or completely arrhythmic phenotypes. This is a unique phenotype that had not previously been observed for mutants at the per locus. An extensive complementation analysis of 3B1-2 chromosome aberrations and per mutant alleles provided no compelling evidence for genetic complexity at the per locus. This is in contrast to the report of Young and Judd (1978). Analysis of both the locomotor-activity and eclosion phenotypes of 3B1-2 chromosome aberrations did not uncover differences in the genetic control of these two rhythms. The clock phenotypes of 3B1-2 chromosome aberrations, the three per mutant alleles, and per ⁺ duplications suggest that mutations at the per locus shorten, lengthen, or eliminate periodicity by respectively increasing, decreasing, or eliminating per activity.     

Chromosomal continuity in the abdominal region of the bithorax complex of Drosophila is not essential for its contribution to metameric identity

We have examined the developmental consequences for larval and imaginal segmental cuticular structure of a chromosomal translocation involving a breakpoint in the abdominal region of the bithorax complex (BX-C). This complex makes an essential contribution to the development of metameric differences in part of the thorax and in all abdominal segments. The breakpoint is proximal to the most distal (iab-7) homeobox, and results in the translocation to the Y chromosome of the Ultrabithorax (Ubx) and abdominal-A (abd-A) domains. The genotype deficient for the distal part of the complex shows normal function for Ubx and abd-A but has a phenotype typical for severe Abd-B mutations. Conversely, the distal fragment retains a segment identity function which must represent a contribution from Abd-B in parasegments 13 and 14; the latter metamere is wild type, indicating that it does not require the contribution of Ubx or abd-A. We also constructed a genotype comprising the proximal fragment of this translocation together with an overlapping distal fragment of the BX-C derived from Df(3R)Ubx109. It therefore contained all sequences of the BX-C though in the abdominal region the abd-A and Abd-B domains were not adjacent to each other in the chromosome. This genotype was phenotypically normal and demonstrates that DNA sequences in the abd-A and Abd-B regions do not require cis-arrangement for their activity.     

Mutations in the gene encoding the adenovirus early region 1B 19,000-molecular-weight tumor antigen cause the degradation of chromosomal DNA

The adenovirus mutant Ad2ts111 has been previously shown to contain a mutation in the early region 2A gene encoding the single-stranded-DNA-binding protein that results in thermolabile replication of virus DNA and a mutation in early region 1 that causes degradation of intracellular DNA. A recombinant virus, Ad2cyt106, has been constructed which contains the Ad2ts111 early region 1 mutation and the wild-type early region 2A gene from adenovirus 5. This virus, like its parent Ad2ts111, has two temperature-independent phenotypes; first, it has the ability to cause an enhanced and unusual cytopathic effect on the host cell (cytocidal [cyt] phenotype) and second, it induces degradation of cell DNA (DNA degradation [deg] phenotype). The mutation responsible for these phenotypes is a single point mutation in the gene encoding the adenovirus early region 1B (E1B) 19,000-molecular-weight (19K) tumor antigen. This mutation causes a change from a serine to an asparagine in the 20th amino acid from the amino terminus of the protein. Three other mutants that affect the E1B 19K protein function have been examined. The mutants Ad2lp5 and Ad5dl337 have both the cytocidal and DNA degradation phenotypes (cyt deg), whereas Ad2lp3 has only the cytocidal phenotype and does not induce degradation of cell DNA (cyt deg+). Thus, the DNA degradation is not caused by the altered cell morphology. Furthermore, the mutant Ad5dl337 does not make any detectable E1B 19K protein product, suggesting that the absence of E1B 19K protein function is responsible for the mutant phenotypes. A fully functional E1B 19K protein is not absolutely required for lytic growth of adenovirus 2 in HeLa cells, and its involvement in transformation of nonpermissive cells to morphological variants is discussed.     

载脂蛋白E基因多态性检测对ACS患者降脂疗效的影响

目的:探究载脂蛋白E(ApoE)基因多态性检测在急性冠脉综合征(ACS)患者降脂治疗的中应用价值。方法:选取2019年2月~2020年6月180例ACS患者,采用随机数字表法分为A、B、C共3组各60例,各组患者均接受ApoE基因多态性检测,并根据Sanger法测序判断ApoE基因表型(E2、E3、E4表型),A组予以瑞舒伐他汀口服(10 mg/d),B组予以瑞舒伐他汀强化治疗(20 mg/d),C组予以瑞舒伐他汀(10 mg/d)+依折麦布(10 mg/d)口服,连续治疗1个月,评价3组各基因表型血脂[总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白(LDL-C)]改善情况、LDL-C达标率,记录药物副反应,所有患者随访1个月,统计心血管不良事件(MACE)发生情况。结果:3组ApoE基因E2、E3、E4表型构成比无显著差异(P>0.05)。治疗后,各组不同ApoE基因表型TC、TG、LDL-C水平均较治疗前下降,且变化率比较差异有统计学意义(P<0.05),表现为E2型>E3型>E4型;其中,3组E2表型TC、TG、LDL-C水平变化率无显著差异(P>0.05);B组、C组E3表型TC、TG、LDL-C水平变化率均显著高于A组(P<0.05),但B组、C组各指标变化率比较差异无统计学意义(P>0.05);3组E4表型TC、TG、LDL-C水平变化率比较差异有统计学意义(P<0.05),且表现为C组>B组>A组。治疗后,A组LDL-C达标率为61.67%,显著低于B组的85.00%、C组的90.00%(P<0.05);其中,3组E2表型LDL-C达标率比较无显著差异(P>0.05),A组E3表型LDL-C达标率显著低于B组、C组(P<0.05),A组、B组E4表型LDL-C达标率低于C组(P<0.05)。治疗期间,仅B组出现1例ALT超出正常上限3倍,停药后可恢复正常。3组MACE发生率比较差异有统计学意义(P<0.05),表现为A组发生率18.33%明显高于B组5.00%、C组3.33%(P<0.05),但3组E2、E4型MACE发生率均无明显差异(P>0.05),而A组E3型MACE发生率高于B组、C组(P<0.05)。结论:ACS患者降脂疗效与ApoE基因表型有关,对E2表型单用瑞舒伐他汀即可取得良好降脂效果,对E3表型强化瑞舒伐他汀或联合依折麦布治疗较单用瑞舒伐他汀均能提高降脂效果,而对E4表型联合依折麦布降脂效果优于单用瑞舒伐他汀或强化治疗。     

Most genes encoding cytoplasmic intermediate filament (IF) proteins of the nematode Caenorhabditis elegans are required in late embryogenesis

Intestinal cells of C. elegans show an unexpectedly high complexity of cytoplasmic intermediate filament (IF) proteins. Of the 11 known IF genes six are coexpressed in the intestine, i.e. genes B2, C1, C2, D1, D2, and E1. Specific antibodies and GFP-promoter constructs show that genes B2, D1, D2, and E1 are exclusively expressed in intestinal cells. Using RNA interference (RNAi) by microinjection at 25 degrees C rather than at 20 degrees C we observe for the first time lethal phenotypes for C1 and D2. RNAi at 25 degrees C also shows that the known A1 phenotype occurs already in the late embryo after microinjection and is also observed by feeding which was not the case at 20 degrees C. Thus, RNAi at 25 degrees C may also be useful for the future analysis of other nematode genes. Finally, we show that triple RNAi at 20 degrees C is necessary for the combinations B2, D1, E1 and B2, D1, D2 to obtain a phenotype. Together with earlier results on genes A1, A2, A3, B1, and C2 RNAi phenotypes are now established for all 11IF genes except for the A4 gene. RNAi phenotypes except for A2 (early larval lethality) and C2 (adult phenotype) relate to the late embryo. We conclude that in C. elegans cytoplasmic IFs are required for tissue integrity including late embryonic stages. This is in strong contrast to the mouse, where ablation of IF genes apparently does not affect the embryo proper.     

Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins.

Infection with adenovirus mutants carrying either point mutations or deletions in the coding region for the 19-kDa E1B gene product (19K protein) causes degradation of host cell and viral DNAs (deg phenotype) and enhanced cytopathic effect (cyt phenotype). Therefore, one function of the E1B 19K protein is to protect nuclear DNA integrity and preserve cytoplasmic architecture during productive adenovirus infection. When placed in the background of a virus incapable of expressing a functional E1A gene product, however, E1B 19K gene mutations do not result in the appearance of the cyt and deg phenotypes. This demonstrated that expression of the E1A proteins was responsible for inducing the appearance of the cyt and deg phenotypes. By constructing a panel of viruses possessing E1A mutations spanning each of the three E1A conserved regions in conjunction with E1B 19K gene mutations, we mapped the induction of the cyt and deg phenotypes to the amino-terminal region of E1A. Viruses that fail to express conserved region 3 (amino acids 140 to 185) and/or 2, (amino acids 121 to 185) or nonconserved sequences between conserved regions 2 and 1 of E1A (amino acids 86 to 120) were still capable of inducing cyt and deg. This indicated that activities associated with these regions, such as transactivation and binding to the product of the retinoblastoma susceptibility gene, were dispensable for induction of E1A-dependent cytotoxic effects. In contrast, deletion of sequences in the amino terminus of E1A (amino acids 22 to 107) resulted in extragenic suppression of the cyt and deg phenotypes. Therefore, a function affected by deletion of amino acids 22 to 86 of E1A is responsible for exerting cytotoxic effects in virally infected cells. Furthermore, transient high-level expression of the E1A region using a cytomegalovirus promoter plasmid expression vector was sufficient to induce the cyt and deg phenotypes, demonstrating that E1A expression alone is sufficient to exert these cytotoxic effects and that other viral gene products are not involved. Finally, placing E1A expression under the control of a strong promoter did not alter the requirement for E1B in the transformation of primary cells. One possibility is that the E1B 19K protein is required to overcome the cytotoxic effects of E1A protein expression and thereby enable primary cells to become transformed.     

The spindle checkpoint kinase bub1 and cyclin e/cdk2 both contribute to the establishment of meiotic metaphase arrest by cytostatic factor

In vertebrate unfertilized eggs, metaphase arrest in Meiosis II is mediated by an activity known as cytostatic factor (CSF). CSF arrest is dependent upon Mos-dependent activation of the MAPK/Rsk pathway, and Rsk activates the spindle checkpoint kinase Bub1, leading to inhibition of the anaphase-promoting complex (APC), an E3 ubiquitin ligase required for the metaphase/anaphase transition. However, it is not known whether Bub1 is required for the establishment of CSF arrest or whether other pathways also contribute. Here, we show that immunodepletion of Bub1 from egg extracts blocks the ability of Mos to establish CSF arrest, and arrest can be restored by the addition of wild-type, but not kinase-dead, Bub1. The appearance of CSF arrest at Meiosis II may result from coexpression of cyclin E/Cdk2 with the MAPK/Bub1 pathway. Cyclin E/Cdk2 was able to cause metaphase arrest in egg extracts even in the absence of Mos and could also inhibit cyclin B degradation in oocytes when expressed at anaphase of Meiosis I. Once it has been established, metaphase arrest can be maintained in the absence of MAPK, Bub1, or cyclin E/Cdk2 activity. Both pathways are independent of each other, but each appears to block activation of the APC, which is required for cyclin B degradation and the metaphase/anaphase transition.     

Genetic identification of adenovirus type 5 genes that influence viral spread

The mechanisms that control cell-to-cell spread of human adenoviruses (Ad) are not well understood. Two early viral proteins, E1B-19K and E3-ADP, appear to have opposing effects since viral mutants that are individually deficient in E1B-19K produce large plaques (G. Chinnadurai, Cell 33:759-766, 1983), while mutants deficient in E3-ADP produce small plaques (A. E. Tollefson et al., J. Virol. 70:2296-2306, 1996) on infected cell monolayers. We have used a genetic strategy to identify different viral genes that influence adenovirus type 5 (Ad5) spread in an epithelial cancer cell line. An Ad5 mutant (dl327; lacking most of the E3 region) with the restricted-spread (small-plaque) phenotype was randomly mutagenized with UV, and 27 large-plaque (lp) mutants were isolated. A combination of analyses of viral proteins and genomic DNA sequences have indicated that 23 mutants contained lesions in the E1B region affecting either 19K or both 19K and 55K proteins. Four other lp mutants contained lesions in early regions E1A and E4, in the early L1 region that codes for the i-leader protein, and in late regions that code for the viral structural proteins, penton base, and fiber. Our results suggest that the requirement of E3-ADP for Ad spread could be readily compensated for by abrogation of the functions of E1B-19K and provide genetic evidence that these two viral proteins influence viral spread in opposing manners. In addition to E1B and E3 proteins, other early and late proteins that regulate viral replication and infectivity also influence lateral viral spread. Our studies have identified novel mutations that could be exploited in designing efficient oncolytic Ad vectors.     

Cytogenetic analysis of variegation suppressors and a dominant temperature-sensitive lethal in region 23–26 of chromosome 2L in Drosophila melanogaster

Three suppressor loci for position-effect variegation, one dominant temperature-sensitive (DTS), three Minute genes, and two recessive visible mutants (ed, tkv) have been cytogenetically localized by using duplications and deficiencies in regions 23-25 of chromosome arm 2L of Drosophila melanogaster. Two of the suppressor loci studied proved to represent haplo-abnormal genes localized in regions 23A6-23F6 and 24E2-25A1, respectively. The third one is a strong triplo-abnormal suppressor mapping in 25F4-26B9 which affects white variegation in wm4h when present in three doses. The l(2)2DTS mutation, which belongs to a group of noncomplementing dominant temperature-sensitive mutations, is localized in the 25A4-B1 region. Furthermore, two Minute genes have been localized in region 24 that are included in Df(2L)M11 and can be separated employing translocation (Y;2)P8 (24E2-4): M(2)LS2 in 24D3-4-24E2-4, and M(2)z in 24E4-5-24F5-7. A third Minute gene (M(2)S1) is localized in 25C3-8-25C9-D1. The usefulness of the isolated chromosomal rearrangements for further genetic studies of region 23-26 is discussed.     

Expression of adenovirus E1B mutant phenotypes is dependent on the host cell and on synthesis of E1A proteins.

Adenovirus mutants containing genetic alterations in the gene encoding the E1B 19,000-molecular-weight (19K) tumor antigen induce the degradation of host cell chromosomal DNA (deg phenotype) and enhanced cytopathic effect (cyt phenotype) after infection of HeLa and KB cells. The deg and cyt phenotypes are a consequence of viral early gene expression in the absence of the E1B 19K protein. The role of the E1A proteins in induction of the cyt and deg phenotypes was investigated by constructing E1A-E1B double mutant viruses. Viruses were constructed to express the individual E1A 13S, 12S, or 9S cDNA genes in the presence of a mutation in the gene encoding the E1B 19K tumor antigen. Expression of either the 13S or 12S E1A proteins in the absence of functional E1B 19K protein produced the deg and cyt phenotypes. In contrast, a virus which expressed exclusively the 9S E1A gene product in the absence of the E1B 19K gene product did not induce the deg and cyt phenotypes, even at high multiplicities of infection. Therefore, both the 13S and 12S E1A gene products could directly or indirectly cause the deg and cyt phenotypes during infection of HeLa cells with an E1B 19K gene mutant virus. Furthermore, the deg phenotype was found to be host cell type specific, occurring in HeLa and KB cells but not in growth-arrested human WI38 cells. These results indicate that expression of the E1A trans-activating and transforming proteins is necessary for the induction of the cyt and deg phenotypes and that host cell factors also play a role.     

Double and triple mutant combinations of bithorax complex of Drosophila

We have constructed double and triple mutant combinations for the Ubx, abd-A and Abd-B genes of the bithorax complex and have examined the homeotic transformations they produce in the larval and adult patterns. Embryos hemizygous for the triple combination exhibit a metameric pattern consisting of parasegments 5-12 being transformed into parasegment 4. In addition, parasegment 13 develops like a mixture of parasegment 3 and 4, and parasegment 14 is abnormal. The same phenotype is displayed by embryos homozygous for DfP9, lacking all the BX-C DNA, >300 kb. This result strongly supports the notion that the BX-C contains only three genes which account for all the developmental functions of the complex. The phenotypes of the different double combinations also support the same view; the Ubx abd-a comthoracic and several abdominal functions. The abd-A Abd-B combination exhibits the same phenotype of DpP10 DfP9, lacking all the abdominal functions except those specific for A1. Our results also indicate that each BX-C gene becomes active autonomously regardless of the presence or functional state of the other BX-C genes.     

An Alu-mediated deletion at Xp11.23 leading to Wiskott-Aldrich syndrome

Wiskott-Aldrich syndrome (WAS) is an X-linked disease characterized by thrombocytopenia, eczema and immunodeficiency of varying severity. The WASP gene, mutations of which are responsible for the phenotype, maps to Xp11.23. We describe here a patient with a large deletion in the Xp11.23 region. The deletion, which totals 15.8 kb, begins downstream of DXS1696 and encompasses 13 kb upstream of WASP and includes the distal and proximal promoters and exons 1-6. Analysis of the 5'-boundary region identified sequences missing in the Human Genome database and, as a result, the normal DNA sequence was revised to include 743 bp of novel sequence (AF466616). The patient's upstream breakpoint was localized to an AluSg element within a highly repetitive DNA region containing other Alu elements. A 26-bp recombinogenic element is located downstream of the 5' breakpoint. A 16-bp sequence just upstream of the 5' breakpoint shares close homology with the sequence that spans the 3' breakpoint in intron 6. A heptanucleotide of unknown origin, CAGGGGG, links the 5' and 3' breakpoints. To our knowledge this is the largest deletion in a WAS patient.     

Genetic systems of red cell blood groups in goats.

Evidence is presented for six genetic systems of goat red cell blood groups. The A system presently consists of one specificity, two alleles, two phenotypes (A1 and no-A1) and appears to be homologous to the A system of sheep. The B system, like its homologue in sheep, is very complex. Fourteen of 21 specificities detected in the present study, i.e. B2, B3, B4, B5, B7, B8, B9, B10, B11, B14, B15, B16, B17 and B20, belong to the B system which involves a large number of phenogroups (31 different B phenogroups identified in 26 sires). Because of their homology with sheep C and R systems, two other genetic systems of goat blood groups are named C and R respectively. Each of the two goat systems is presently a one blood group specificity, two phenotype (C12 and no-C12; R and no-R detectable on the red cells) two allele system. Two specificities, namely E6 and E18, belong to a genetic system called E in which four phenotypes are under the control of two alleles codominant and one recessive at a single locus. The F system involves but a single pair of alleles and two phenotypes (F19 and no-F19). Because of its low frequency in the goats tested, the X13 specificity remains unassigned.     

Differential regulation of cyclin B1 degradation between the first and second meiotic divisions of bovine oocytes

During mammalian oocyte maturation, two consecutive meiotic divisions are required to form a haploid gamete. For each meiotic division, oocytes must transfer from metaphase to anaphase, but maturation promoting factor (cyclin-dependent kinase 1/cyclin B1) activity would keep the oocytes at metaphase. Therefore, inactivation of maturation promoting factor is needed to finish the transition and complete both these divisions; this is provided through anaphase-promoting complex/cyclosome-dependent degradation of cyclin B1. The objective of this study was to examine meiotic divisions in bovine oocytes after expression of a full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion, coupled with the fluorochrome Venus, by microinjecting their complementary RNA (cRNA). Overexpression of full-length cyclin B1-Venus inhibited homologue disjunction and first polar body formation in maturing oocytes (control 70% vs. overexpression 16%; P < 0.05). However at the same levels of expression, it did not block second meiotic metaphase and cleavage of eggs after parthenogenetic activation (control: 82% pronuclei and 79% cleaved; overexpression: 91% pronuclei and 89% cleaved). The full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion caused metaphase arrest in both meiotic divisions, whereas degradation of securin was unaffected. Roscovitine, a potent cyclin-dependent kinase 1 (CDK1) inhibitor, overcame this metaphase arrest in maturing oocytes at 140 μM, but higher doses (200 μM) were needed to overcome arrest in eggs. In conclusion, because metaphase I (MI) blocked by nondegradable cyclin B1 was distinct from metaphase II (MII) in their different sensitivities to trigger CDK1 inactivation, we concluded that mechanisms of MI arrest differed from MII arrest.