Autoregulatory Functioning of a Drosophila Gene Product That Establishes and Maintains the Sexually Determined State

Sxl appears to head a regulatory gene hierarchy that controls Drosophila sexual dimorphism in response to the X chromosome/autosome balance. Only XXAA cells normally have Sxl⁺ activity. It maintains both the female morphogenetic sequence and a level of X-linked dosage-compensated gene expression compatible with diplo-X cell survival. In the absence of this activity, male sexual development and dosage-compensated gene hyperactivation ensure. Loss-of-function Sxl mutations generally have female-specific lethal effects caused by upsets in dosage compensation. New female-viable Sxl mutant alleles and combinations which lack Sxl's female sex determination function, yet still provide sufficient dosage compensation function for diplo-X survival, are described here. Consequently, such mutants cause genotypic females to develop as phenotypic males. Some of these sex-transforming Sxl mutants do not require the maternally produced da⁺ activity that is normally essential for the functioning of zygotic Sxl alleles. In this paper, products of these unusual alleles are shown to act in trans to induce the expression of zygotic Sxl⁺ alleles that would otherwise be unable to function due to a lack of maternal da⁺ activity. This result indicates a third function for Sxl⁺ product: a positive autoregulatory role. Controls for the autoregulation experiments demonstrated the sex-trans-forming epigenetic effect of the da mutation for the first time in diploids. In these experiments the female-specific zygotic lethal effects that normally would have accompanied loss of maternal da⁺ activity were suppressed by mutations known to block dosage-compensation gene hyperactivation—the autosomal, male-specific lethals. Three types of abnormal sexual phenotypes were produced in the experiments described here, each with important implications for the mechanism of sex determination: (1) a true intersex phenotype produced by one particular Sxl allele shows that Sxl⁺ must be involved in the cellular response to the X/A balance rather than in its establishment; (2) a maternally induced, female-sterile phenotype indicates that either the process of autoregulation or the mutants used to demonstrate it are tissue specific and (3) a mosaic intersexual phenotype whose character implies that the Sxl⁺ activity level is set early in development, both by the da ⁺-mediated X/A balance signal and by autoregulation, and is maintained subsequently in a cell autonomous fashion, independent of the initiating X/A balance signal. Thus, this study supports the view that sex determination is truly determinative in the standard developmental sense, and that Sxl is the carrier of the sexually determined state.     

Copy Number Variation at the APOL1 Locus

Two coding variants in the APOL1 gene (G1 and G2) explain most of the high rate of kidney disease in African Americans. APOL1-associated kidney disease risk inheritance follows an autosomal recessive pattern: The relative risk of kidney disease associated with inheritance of two high-risk variants is 7–30 fold, depending on the specific kidney phenotype. We wished to determine if the variability in phenotype might in part reflect structural differences in APOL1 gene. We analyzed sequence coverage from 1000 Genomes Project Phase 3 samples as well as exome sequencing data from African American kidney disease cases for copy number variation. 8 samples sequenced in the 1000 Genomes Project showed increased coverage over a ~100kb region that includes APOL2, APOL1 and part of MYH9, suggesting the presence of APOL1 copy number greater than 2. We reasoned that such duplications should be enriched in apparent G1 heterozygotes with kidney disease. Using a PCR-based assay, we observed the presence of this duplication in additional samples from apparent G0G1 or G0G2 individuals. The frequency of this APOL1 duplication was compared among cases (n = 123) and controls (n = 255) with apparent G0G1 heterozygosity. The presence of APOL1 duplication was observed in 4.06% of cases and 0.78% controls, preliminary evidence that this APOL1 duplication may alter susceptibility to kidney disease (p = 0.03). Taqman-based copy number assays confirmed the presence of 3 APOL1 copies in individuals positive for this specific duplication by PCR assay, but also identified a small number of individuals with additional APOL1 copies of presumably different structure. These observations motivate further studies to better assess the contribution of APOL1 copy number on kidney disease risk and on APOL1 function. Investigators and clinicians genotyping APOL1 should also consider whether the particular genotyping platform used is subject to technical errors when more than two copies of APOL1 are present.     

Reversion at the HIS1 Locus of Yeast

The his1 gene (chromosome V) of Saccharomyces cerevisiae specifies phosphoribosyl transferase (E.C.2.4.2.17), the first enzyme of histidine biosynthesis. This hexameric enzyme has both catalytic and regulatory functions. The spontaneous reversion rates of seven his1 mutations were studied. The reversion rates of the alleles at the proximal end of the locus (relative to the centromere) were about 50-fold higher than distal alleles. Spontaneous reversion to prototrophy was studied in diploids homoallelic for each of the seven his1 mutations. Based on tetrad analysis, the prototrophy revertants could be assigned to three classes: (1) revertant tetrads that carried a prototrophic allele indistinguishable from wild type; (2) revertant tetrads that carried a prototrophic allele characterized by histidine excretion and feedback resistance; and (3) revertant tetrads that did not contain a prototrophic spore, but rather a newly derived allele that complemented the original allele intragenically. Four of the seven his1 mutations produced the excretor revertant class, and two mutations produced the complementer revertant class. The significance of these findings to our understanding of gene organization and the catalytic and regulatory functions of gene products are discussed.     

Regulatory Mutants at the his1 Locus of Yeast

The his1 gene in Saccharomyces cerevisiae codes for phosphoribosyl transferase, an allosteric enzyme that catalyzes the initial step in histidine biosynthesis. Mutants that specifically alter the feedback regulatory function were isolated by selecting his1 prototrophic revertants that overproduce and excrete histidine. The prototrophs were obtained from diploids homoallelic for his1--7 and heterozygous for the flanking markers thr3 and arg6. Among six independently derived mutant isolates, three distinct levels of histidine excretion were detected. The mutants were shown to be second-site alterations mapping at the his1 locus by recovery of the original auxotrophic parental alleles. The double mutants, HIS1--7e, are dominant with respect to catalytic function but recessive in regulatory function. When removed from this his1--7 background, the mutant regulatory site (HIS1-e) still confers prototrophy but not histidine excretion. To yield the excretion phenotype, the primary and altered secondary sites are required in cis array. Differences in histidine excretion levels correlate with resistance to the histidine analogue, triazoalanine.     

The Conserved L5 Loop Establishes the Pre-Powerstroke Conformation of the Kinesin-5 Motor, Eg5

Kinesin superfamily motor proteins contain a structurally conserved loop near the ATP binding site, termed L5. The function of L5 is unknown, although several drug inhibitors of the mitotic kinesin Eg5 bind to L5. We used electron paramagnetic resonance spectroscopy (EPR) to investigate the function of L5 in Eg5. We site-specifically attached EPR probes to ADP, L5, and the neck linker element that docks along the enzymatic head to drive forward motility on microtubules (MTs). Nucleotide-dependent spectral mobility shifts occurred in all of these structural elements, suggesting that they undergo coupled conformational changes. These spectral shifts were altered by deletion of L5 or addition of S-trityl-l-cysteine (STLC), an allosteric inhibitor that binds to L5. In particular, EPR probes attached to the neck linker of MT-bound Eg5 shifted to a more immobilized component in the nucleotide-free state relative to the ADP-bound state, consistent with the neck linker docking upon ADP release. In contrast, after L5 deletion or STLC addition, EPR spectra were highly immobilized in all nucleotide states. We conclude that L5 undergoes a conformational change that enables Eg5 to bind to MTs in a pre-powerstroke state. Deletion or inhibition of L5 with the small-molecule inhibitor STLC blocks this pre-powerstroke state, forcing the Eg5 neck linker to dock regardless of the nucleotide state.     

Novel Interallelic Complementation at the his1 Locus of Yeast

In yeast, 17 histidine-requiring mutants derived from and interallelically complementary to his1-7 were analyzed. The genetic basis of the complementation response was elucidated by mitotic and meiotic gene conversion. Each allele probably carries an unaltered 7-site mutation and a unique second-site alteration. The second-site alterations appear to be clustered within the proximal and distal segments of the his1 structural gene. Models of intraalelic complementation are reviewed in light of the unique complementational response between a single-site mutant and a double mutant including the identical altered base sequence.     

Frequency-Dependent Selection at the PGM-1 Locus of DROSOPHILA PSEUDOOBSCURA

Frequency-dependent fitness was studied at the Pgm-1 locus of Drosophila pseudoobscura with respect to two fitness components: rate of development and larva-to-adult survival. The Pgm-1 locus is very polymorphic with only two alleles, Pgm-1¹⁰⁰ and Pgm-1¹⁰⁴, occurring at high frequencies. For each of these two alleles, 20 homozygous strains were obtained from a sample of 1,140 wild-inseminated females. First-instar larvae of the two genotypes were combined in a set of eight different frequencies: 0.0, 0.10, 0.25, 0.40, 0.60, 0.75, 0.90, and 1.0. Frequency-dependent fitness effects were observed for the two survival-related fitness components examined: larvae of the less common genotype develop faster and have a higher probability of survival than larvae of the more common genotype. The rate of survival at intermediate genotypic frequencies is similar to that in pure cultures. If selection acted solely as frequency-dependent effects on survival-related components of fitness, the equilibrium frequency of the Pgm-1¹⁰⁰ allele would be 0.615 for a two-genotype system, which fits an observed frequency range for this allele in nature between 0.55 and 0.71. Experimentally created linkage disequilibrium was excluded from the experiment by using a large number of independent strains. It is nevertheless possible that the frequency-dependent selection may not affect the Pgm-1 locus per se, but may reflect a linkage disequilibrium present in the natural population. Even if this were the case, the frequency-dependent selection could affect the frequency of the Pgm-1 alleles in nature.     

Analysis of Yeast Retrotransposon Ty Insertions at the Can1 Locus

The target site distribution for 55 independent Ty insertions that inactivate the function of the Saccharomyces cerevisiae CAN1 gene is reported. Under some selection conditions Ty elements inserted preferentially into the promoter and exhibited an orientation bias. In contrast, under other conditions no insertions were detected in the promoter region and transposition appeared to occur randomly throughout the CAN1 coding sequence. These results show that the target site distribution for Ty insertions may be a function of the selection conditions.     

HLA－DRB1基因位点多态性的PCR－RFLP分析

设计并建立一套适合国内应用的改良ＰＣＲ－ＲＦＬＲ方法,分５组特异性扩增ＤＮＡ样品,随后进行酶切定型分析,准确检测了编码ＤＲ抗原特异性的ＨＬＡ－ＤＲＢ１基因位点的多态性,该法采用分组扩增,不发生与其它ＤＲＢ位点等位基因的交叉扩增,不仅适合纯合子的区分而且可以清楚准确地检测杂合子样品,已报道过的ＤＲＢ１位点编码的特异性组合都可以通过这个方法得到准确分析。所使用的Ⅱ类限制性内切酶均价格便宜、易购。     

Chromatin compaction at the mononucleosome level

Using a previously described FRET technique, we measured the distance between the ends of DNA fragments on which nucleosomes were reconstituted from recombinant and native histones. This distance was analyzed in its dependence on the DNA fragment length, concentration of mono- and divalent counterions, presence of linker histone H1, and histone modifications. We found that the linker DNA arms do not cross under all conditions studied but diverge slightly as they leave the histone core surface. Histone H1 leads to a global approach of the linker DNA arms, confirming the notion of a "stem structure". Increasing salt concentration also leads to an approach of the linker DNAs. To study the effect of acetylation, we compared chemically acetylated recombinant histones with histones prepared from HeLa cells, characterizing the sites of acetylation by mass spectroscopy. Nucleosomes from chemically acetylated histones have few modifications in the core domain and form nucleosomes normally. Acetylating all histones or selectively only H3 causes an opening of the nucleosome structure, indicated by the larger distances between the linker DNA ends. Selective acetylation of H4 distances the linker ends for short fragments but causes them to approach each other for fragments longer than 180 bp.     

Genome-wide Chromatin Interactions of the Nanog Locus in Pluripotency,Differentiation, and Reprogramming

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大鼠老化过程大脑皮层及小脑神经元染色质构象分析

 本文在前文~[2]的基础上进一步以MCN和DNaseⅠ为探针研究大鼠脑神经元终末分化后不同生理时期染色质构象,结果表明:MCN酶解DNA产物PAGE显示脑老化过程大脑皮层及小脑神经元染色质核小体单体DNA分别保持在176bp和215bp水平,核小体连接DNA长度存在组织差异,但不受老化影响;<2>DNaseⅠ酶解DNA产物PAGE显示各年龄组大脑皮层及小脑神经元染色质DNA存在10bp间隔重复结构和相同的泳动区带分布特征,提示脑老化中染色质具有稳定的B型双螺旋结构和一致的螺线管卷曲形式。染色质DNaseⅠ降解率随年龄增加而降低,提示老化导致活性染色质区域减少,老化过程脑神经元染色质构象改变成为其转录功能减退的结构基础。