Proposed role of W chromosome inactivation and the absence of dosage compensation in avian sex determination

Three features of avian sex chromosomes-female heterogamety (ZZ male, ZW female), the apparently inactive state of the W chromosome, and dose-dependent expression of Z-linked genes-are examined in regard to their possible relation to sex determination. It is proposed that the W chromosome is facultatively heterochromatic and that the Z and W chromosomes carry one or more homologous sex-determination genes. The absence of dosage compensation in ZZ embryos, and W inactivation in ZW embryos, would then bring about a 2n(ZZ)-n(ZW) inequality in the effective copy number of such genes. The absence of dosage compensation of Z-linked genes in ZZ embryos is viewed as a means by which two copies of Z-W homologous sex determination genes are kept active to meet the requirements of testis determination. W inactivation may promote ovarian development by reducing the effective copy number of these genes from 2n to n. If there is a W-specific gene for femaleness, spread of heterochromatization to this gene in cells forming the right gonadal primordium may explain the latter's normally undifferentiated state; reversal of heterochromatization may similarly explain the development of the right gonad into a testis following left ovariectomy

The evolution of genomic imprinting

We explore three possible pathways for the evolution of genomic imprinting. (1) Imprinting may be advantageous in itself when imprinted and unimprinted alleles of a locus confer different phenotypes. If a segment of DNA is imprinted in the gametes of one sex but not in those of the other, it might lead to effects correlated with sexual dimorphism. More fundamentally, in certain organisms, sex determination might have evolved because of imprinting. When imprinting leads to chromosome elimination or inactivation and occurs in some embryos but not in others, two classes of embryos, differing in the number of functional gene copies, would result. A model for sex determination based on inequality in the actual or effective copy-number of particular noncoding, regulatory sequences of DNA has been proposed (Chandra, Proc. natn. Acad. Sci. U.S.A. 82. 1165-1169 and 6947-6949, 1985). Maternal control of offspring sex is another possible consequence of imprinting; this would indicate a potential role for imprinting in sex ratio evolution. (2) Genes responsible for imprinting may have pleiotropic effects and they may have been selected for reasons other than their imprinting ability. Lack of evidence precludes further consideration of this possibility. (3) Imprinting could have co-evolved with other traits. For instance, gamete-specific imprinting could lead to a lowered fitness of androgenetic or gynogenetic diploids relative to the fitness of 'normal' diploids. This in turn would reinforce the evolution of anisogamy. The reversibility of imprinting raises the possibility of occasional incomplete or improper erasure. If the site of imprinting is the egg - as appears to be the case with the human X (Chandra and Brown, Nature 253. 165-168, 1975) - either improper imprinting or improper erasure could lead to unusual patterns of inheritance (as in the fragile-X syndrome) or fitness effects skipping generations

The inactive X chromosome in the human female is enriched in 5-methylcytosine to an unusual degree and appears to contain more of this modified nucleotide than the remainder of the genome

By employing a procedure that combines ELISA and photoacoustic spectroscopy, we have examined the content of 5-methylcytosine (m5C) in DNA of individuals who differed from one another in the number of X chromosomes in their genomes. The results show that the human inactive X chromosome (Xi) contains very high amounts of this modified nucleotide. We estimate that in the 46,XX female there is more m5C in Xi (~3.6 × 107) than in all the remaining chromosomes put together (~2.1 × 107). Our results also suggest that nearly one-fifth of all cytosines in Xi are methylated and that, in addition to CpG methylation, there is extensive non-CpG methylation as well

A rapid and gentle method for the salt extraction of chromatin core histones H2A, H2B, H3 and H4 from rat liver nuclei

A complex of histones H2A, H2B, H3 and H4 has been isolated from purified rat liver nuclei by a method which is both gentle and rapid. Nuclei were homogenised in 0.25 I sucrose and the residual nuclear material obtained after centrifligation was adsorbed on calcium phosphate gel. After removing histone H1 from the adsorbed material by washing with 1M NaCl in 25 mM sodium phosphate buffer, pH 6.0, histones H2A, H2B, H3 and H4 were eluted together, with 2 I NaCl in 25 mM sodium phosphate buffer, pH 7.0. The core histones so obtained migrated as a single sharp band on polyacrylamide gel electrophoresis under non-denaturing conditions. Fractionation of the freshly prepared core histones on a Sephadex G-100 column yielded two major protein peaks. The peak having the larger elution volume contained histones H2A and H2B in equal amounts while the peak with the smaller elution volume contained all the four histones. Histones H3 and H4 were present in larger proportions in the second peak

Requirement of flex (female lethal on X) in the development of the female germ line of Drosophila melanogaster

Drosophila melanogaster females homozygous for flex, an X- linked recessive mutation, do not survive. Hemizygous males are unaffected. Homozygous embryos appear to lack SXL, the product of the Sex-lethal (Sxl) gene, apparently as a result of disruption of Sxl splicing. It is known that both Sxl and its somatic splicing regulators [snf and fl(2)d] also function in the development of the female germ line. For this reason, we investigated the role of flex in the germ line by generating flex/flex clones in flex/+ females. Females carrying such clones in their germ lines do not lay eggs whereas females carrying flex eggs lay viable eggs. Additionally, DAPI staining of ovarioles showed that diploid germ cells that are homozygous mutant for flex do not complete oogenesis. These results indicate that the flex+ gene product may be required for the development of the female germ line

Dosage compensation and sex determination in Drosophila: mechanism of measurement of the X/A ratio+

We propose a molecular mechanism for the intra-cellular measurement of the ratio of the number of X chromosomes to the number of sets of autosomes, a process central to both sex determination and dosage compensation in Drosophila melanogaster. In addition to the two loci, da and Sxl, which have been shown by Cline and others to be involved in these processes, we postulate two other loci, one autosomal (ω) and the other, X-linked (π). The product of the autosomal locusda stimulates ω and initiates synthesis of a limited quantity of repressor. Sxl and π, both of which are X-linked, compete for this repressor as well as for RNA polymerase. It is assumed that Sxl has lower affinity than π for repressor as well as polymerase and that the binding of polymerase to one of these sites modulates the binding affinity of the other site for the enzyme. It can be shown that as a result of these postulated interactions transcription from the Sxl site is proportional to the X/A ratio such that the levels of Sxl+ product are low in males, high in females and intermediate in the intersexes. If, as proposed by Cline, the Sxl- product is an inhibitor of X chromosome activity, this would result in dosage compensation. The model leads to the conclusion that high levels of Sxl+ product promote a female phenotype and low levels, a male phenotype. One interesting consequence of the assumptions on which the model is based is that the level of Sxl+ product in the cell, when examined as a function of increasing repressor concentration, first goes up and then decreases, yielding a bell-shaped curve. This feature of the model provides an explanation for some of the remarkable interactions among mutants at the Sxl, da and mle loci and leads to several predictions. The proposed mechanism may also have relevance to certain other problems, such as size regulation during development, which seem to involve measurement of ratios at the cellular level

A power-efficient thermocycler based on induction heating for DNA amplification by polymerase chain reaction

We have built a thermocycler based on the principles of induction heating for polymerase chain reaction (PCR) of target sequences in DNA samples of interest. The cycler has an average heating rate of ~0.8°C/s and a cooling rate of ~0.5°C/s, and typically takes ~4 h to complete a 40-cycle PCR protocol. It is power-efficient (~6 W per reaction tube), micro-processor controlled, and can be adapted for battery operation. Using this instrument, we have successfully amplified a 350 bp segment from a plasmid and SRY, the human sex determining gene, which occurs as a single-copy sequence in genomic DNA of human males. The PCR products from this thermocycler are comparable to those obtained by the use of commercially available machines. Its easy front-end operation, low-power design, portability and low cost makes it suitable for diagnostic field applications of PCR

The mealybug chromosome system I: unusual methylated bases and dinucleotides in DNA of a Planococcus species

The methylation status of the nuclear DNA from a mealybug, a Planococcus species, has been studied. Analysis of this DNA by High Performance Liquid Chromatography and Thin Layer Chromatography revealed the presence of significant amounts of 5--methylcytosine. Since analysis of DNA methylation using the Msp I/Hpa II system showed only minor differences in susceptibility of the DNA to the two enzymes, it seemed possible that 5-methylcytosine (5mC) occurred adjacent to other nucleotides in addition to its usual position, next to guanosine. This was verified by dinucleotide analysis of DNA labelledin vitro by nick translation. These data show that the total amount of 5-methylcytosine in this DNA is slightly over 2.3 mol %, of which 0.61% occurs as the dinucleotide 5mCpG, 0.68% as 5mCpA, 0.59% as 5mCpT and 0.45% as 5mCpC. 5mCpG represents approximately 3.3% of all CpG dinucleotides. The experimental procedure would not have permitted the detection of 5mCp5mC, if it occurs in this system. Unusually high amounts of 6-methyladenine (approximately 4 mol %) and 7-methylguanine (approximately 2 mol %) were also detected, 6-methyladenine and 7-methylguanine occurred adjacent to all four nucleotides. The total G+C content was 33.7% as calculated from dinucleotide data and 32.9% as determined from melting profiles

Efficacy of surface error corrections to density functional theory calculations of vacancy formation energy in transition metals

We calculate properties like equilibrium lattice parameter, bulk modulus and monovacancy formation energy for nickel (Ni), iron (Fe) and chromium (Cr) using Kohn-Sham density functional theory (DFT). We compare relative performance of local density approximation (LDA) and generalized gradient approximation (GGA) for predicting such physical properties for these metals. We also make a relative study between two different flavors of GGA exchange correlation functional, namely, PW91 and PBE. These calculations show that there is a discrepancy between DFT calculations and experimental data. In order to understand this discrepancy in the calculation of vacancy formation energy, we introduce a correction for the surface intrinsic error corresponding to an exchange correlation functional using the scheme implemented by Mattsson et al. [Phys. Rev. B 73, 195123 (2006)] and compare the effectiveness of the correction scheme for Al and the 3d-transition metals.Comment: 29 pages, 3 figures, 5 tables, Published in J. Phys: Cond. Matt. vol.22 (2010) 34550