DNA-protein interactions in the Drosophila virilis mitochondrial chromosome.

The location of proteins on the mitochondrial DNA (mtDNA) of Drosophila virilis was investigated by Me3 psoralen photoreaction of mitochondria isolated from embryos. After photoreaction the mtDNA was purified and the pattern of DNA cross-linking was determined by electron microscopy of the DNA under totally denaturing conditions. The transcribed regions of the mtDNA molecule contained some uncross-linked regions, but such regions were infrequent and randomly distributed. In contrast, the A + T-rich region around the origin of replication of the mtDNA was usually protected from psoralen cross-linking. The data were best fit by two protected sites, each approximately 400 base pairs, compared to the four 400 base pair sites observed in the equivalent region of D. melanogaster mtDNA [Potter et al. (1980) Proc. Nat. Acad. Sci. USA 77, 4118-4122]. Thus this region of the mtDNA appears to be involved in a DNA-protein structure that is highly conserved even though the DNA sequence has diverged rapidly relative to protein-coding sequences.     

Fragile X chromosome and chromosome condensation

The effect of chromosome condensation on the frequency of expression of the fragile X chromosome was examined. Chromosome decondensation substances were tested for their ability to elicit expression or improve frequencies of expression of the fragile X chromosome in five patients. The substances tested included the AT specific DNA ligands ethidium bromide, Hoechst 33258, and netropsin, and the GC specific substances actinomycin D and olivomycin. Under culture conditions appropriate for eliciting fragile X expression none of the decondensation compounds studied significantly altered frequencies of expression, nor did any of the substances elicit fragile X expression under conditions that normally suppress fragile X expression. The fragile X was found to be more frequently evident in less condensed chromosome preparations from fibroblasts. The implications of these findings with respect to the nature of fragile sites are discussed.     

Genomic relationships based on X chromosome markers and accuracy of genomic predictions with and without X chromosome markers

Background

Although the X chromosome is the second largest bovine chromosome, markers on the X chromosome are not used for genomic prediction in some countries and populations. In this study, we presented a method for computing genomic relationships using X chromosome markers, investigated the accuracy of imputation from a low density (7K) to the 54K SNP (single nucleotide polymorphism) panel, and compared the accuracy of genomic prediction with and without using X chromosome markers.

Methods

The impact of considering X chromosome markers on prediction accuracy was assessed using data from Nordic Holstein bulls and different sets of SNPs: (a) the 54K SNPs for reference and test animals, (b) SNPs imputed from the 7K to the 54K SNP panel for test animals, (c) SNPs imputed from the 7K to the 54K panel for half of the reference animals, and (d) the 7K SNP panel for all animals. Beagle and Findhap were used for imputation. GBLUP (genomic best linear unbiased prediction) models with or without X chromosome markers and with or without a residual polygenic effect were used to predict genomic breeding values for 15 traits.

Results

Averaged over the two imputation datasets, correlation coefficients between imputed and true genotypes for autosomal markers, pseudo-autosomal markers, and X-specific markers were 0.971, 0.831 and 0.935 when using Findhap, and 0.983, 0.856 and 0.937 when using Beagle. Estimated reliabilities of genomic predictions based on the imputed datasets using Findhap or Beagle were very close to those using the real 54K data. Genomic prediction using all markers gave slightly higher reliabilities than predictions without X chromosome markers. Based on our data which included only bulls, using a G matrix that accounted for sex-linked relationships did not improve prediction, compared with a G matrix that did not account for sex-linked relationships. A model that included a polygenic effect did not recover the loss of prediction accuracy from exclusion of X chromosome markers.

Conclusions

The results from this study suggest that markers on the X chromosome contribute to accuracy of genomic predictions and should be used for routine genomic evaluation.     

Mouse X chromosome

Overall, the probe map fromDXWas70 toAmg encompasses 72 cM and includes 103 loci. Eight of these have been designated reference loci (see Table 2 and previous section) on account of their wide usage that would enable the cross reference of independent maps created in different laboratories. Reference loci are to be readily available and easily used probes for Southern hybridization. By and large, they will be STSs, regeneratable by PCR, and correspond to a known gene. In addition, on the mouse X Chr, there is a reference locus from each of the known conserved linkage groups found between the mouse and human X Chrs. All the loci, barDXWas31, represent conserved sequences. Committee Members: D. Adler, P.J. Barnard, Y. Boyd, N. Brockdorff, J. Derry, C. Disteche, C. Faust, M.F. Lyon, S. Rastan, M. Seldin and L. Siracusa.     

The X chromosome and fragile X mental retardation

Fragile X syndrome represents the most common inherited cause of mental retardation. It is caused by a stretch of CGG repeats within the fragile X gene, which increases in length as it is transmitted from generation to generation. Once the repeat exceeds a threshold length, no protein is produced, resulting in the fragile X phenotype. Both X chromosome inactivation and inactivation of the FMR1 gene are the result of methylation. X inactivation occurs earlier than inactivation of the FMR1 gene. The instability to a full mutation is dependent on the sex of the transmitting parent and occurs only from mother to child. For most X-chromosomal diseases, female carriers do not express the phenotype. A clear exception is fragile X syndrome. It is clear that more than 50% of the neurons have to express the protein to ensure a normal phenotype in females. This means that a normal phenotype in female carriers of a full mutation is accompanied by a distortion of the normal distribution of X inactivation.     

Methylation and the X chromosome

Recent approaches towards an understanding of the molecular basis of X-chromosome inactivation in mammals suggest that regulation is due to multiple events including DNA methylation.     

X chromosome loss and ageing

The detection of a low level 45,X cell line during routine cytogenetic analysis in an adult female can be difficult to interpret. In the absence of recent information regarding loss of the X chromosome and ageing, we undertook a prospective study. A total of 19,650 cells from 655 females aged from birth to 80 years were screened cytogenetically. The frequency of X chromosome loss ranged from 0.07% at age <16 years to 7.3% at >65 years of age and showed a highly significant quadratic relationship between X chromosome loss and ageing (P < or = 0.00001). We have produced a graphic representation that provides a minimum baseline age-related rate of X chromosome loss. This should assist diagnostic cytogenetics laboratories to determine the significance of 45,X cell lines detected in women of all ages. We also compared the frequency of 45,X cells in women referred with at least one spontaneous abortion with those referred for other reasons and found no significant difference. Thus, in our population, an excess of 45,X cells is not associated with pregnancy loss.     

Asymmetrical orientation of phospholipids and their interactions with marker enzymes in pig heart mitochondrial inner membrane

The transverse distribution of phospholipids and their interactions with marker enzymes were investigated in pig heart mitoplasts and inverted vesicles, using phospholipase A₂ from N. naja venom and chemical labeling with TNBS and FDNB. Morphological integrity was checked by freeze-fracturing. Fifty percent of phosphatidylcholine was hydrolyzed in mitoplasts as well as in inverted vesicles, suggesting an even distribution of this phospholipid on the two halves of the inner membrane; however, the fatty acid distribution did not appear the same in the two membrane fractions. Cardiolipin is exclusively hydrolyzed in inverted vesicles proving its location on the inner face of the inner membrane. The results obtained from phospholipase hydrolysis and TNBS labeling suggest that three different pools of phosphatidylethanolamine occur in the membrane: a first pool—about 50–60% of the total membrane phosphatidylethanolamine–is quickly accessible from the two sides of the membrane, a second pool—about 20–30% is slowly available, and finally 20–30% are buried within the membrane and inaccessible to the phospholipase and the probe. The cytochrome c oxidase activity increased in mitoplasts with the phospholipase attack suggesting a better accessibility of added cytochrome c after the attack. The rotenone-sensitive NADH-cytochrome c reductase was activated in mitoplasts but completely inactivated in inverted vesicles by the attack; the addition of cardiolipin liposomes restored the latter activity. The soluble matricial malate dehydrogenase was released, but the particulate form of this enzyme, strongly associated to the membrane, was detached only after attack of inverted vesicles.     

The grasshopper X chromosome

The X chromosome can be identified with the light microscope throughout all stages of the gonial cell cycle (including interphase) in the grasshopper Brachystola magna. At gonial mitotic stages the X chromosome gives the appearance of being undercondensed or negatively heteropycnotic. At interphase the X projects out from the body of the nucleus. — Examination with the electron microscope reveals that the X is compartmentalized at least two gonial cell cycles prior to the entry of the cells into meiotic prophase. The membrane layers that envelope the X chromatin at interphase remain associated with the X chromosome throughout gonial mitotic stages providing the ultrastructural basis for the apparent negative heteropycnosis observed with the light microscope. — The X chromosome is inactive in RNA synthesis during gonial mitotic stages but is hyperactive in RNA synthesis when compared to autosomes at gonial interphase. — X chromosome condensation which reaches its maximum at premieotic interphase is initiated at or prior to the pre-pentultimate gonial division.     

DNA-protein interactions and chromosome banding

Pancreatic DNase I was used as a probe to study DNA-protein interactions in condensed and extended chromatin fractions isolated from Chinese hamster liver, and in human lymphocyte and mouse L cell metaphase chromosomes in situ. By studying the rate of digestion of chromatin DNA by DNase, we have previously shown that DNA in extended chromatin is more sensitive to DNase digestion than that in condensed chromatin. In the current investigation, we have examined whether this differential sensitivity of the chromatin fractions to DNase is due to differences in protein binding to DNA or differences in the degree of chromatin condensation. By “decondensing” the condensed chromatin and comparing its rate of digestion to that of untreated condensed and extended chromatin, it was found that differences in the degree of binding of proteins to DNA rather than the degree of condensation of the chromatin primarily determines the sensitivity of each fraction to DNase. Extraction of the various classes of chromosomal proteins, followed by DNase digestion of the residual chromatin revealed that both the histone and non-histone proteins protect the DNA in the chromatin fractions from DNase attack; however, the more tightly associated non-histones appear to be specifically responsible for the differential sensitivity of the chromatin fractions to DNase digestion. These non-histones may be more tightly associated with the DNA in condensed than in extended chromatin, thereby protecting the DNA in condensed chromatin against DNase attack to a greater extent than that in extended chromatin. When metaphase chromosomes were briefly digested with DNase in situ and subsequently stained with Feulgen reagent, incontrovertible C-banding and some G-banding was obtained. This DNaseinduced banding demonstrates that the DNA in C-band and possibly G-band regions is less accessible to DNase than that in the interband regions, and our biochemical data suggest that this differential accessibility is caused by differential DNA-protein binding such that the non-histones are more tightly coupled to the DNA in the G- and C-band regions than they are in the interbands. Differences in the binding of non-histones to DNA in different segments of the metaphase chromosome may be involved in the mechanism of G- and C-banding.     

Assessment of X bends in patients with atypical X chromosome phenotypes.

Several patients with X chromosome structural abnormalities have been more severely affected clinically than expected. Since bends at Xq13-21 have been associated with inactivation, the authors scored bends retrospectively in 62 patients with X chromosome aneuploidy and 21 cases with structural abnormalities of the X chromosome. They found that patients with 2 X inactivation sites where one X was structurally abnormal had significantly fewer cells with X bends than normal 46,XX. In addition, these patients also showed X bends on the normal X more often than would be expected if non-random X inactivation of the abnormal X chromosome was occurring. Five of the 6 patients with a short or long arm deletion or paracentric inversion of Xq were mentally retarded or had other congenital anomalies not usually associated with Turner syndrome. This suggests to them that these clinical findings may be related to interference with X inactivation patterns in cells with a structurally abnormal X chromosome.     

Metal interactions with beef heart mitochondrial ATPase

Atomic absorption and electron paramagnetic resonance spectroscopy were used to study the metal binding sites of beef heart mitochondrial ATPase (F1). Quantitative and qualitative properties of these sites are described. Two different separation techniques were able to distinguish two very tight sites from one tight (easily exchangeable) metal binding site on F1. Of these sites, two are specific for magnesium while one can be substituted with Mn2+, Co2+, or Zn2+. When MgAMP-PNP was incubated with F1, a fourth metal was bound to the enzyme. The carboxyl group modified by dicyclohexylcarbodiimide is shown not to be involved in binding of any of the tightly bound metals. Qualitative properties of the metal binding sites using the Mn2+-enzyme complex as a probe were ascertained using EPR at pH 6.8 and 8.0. CrATP and Mn2+ appear to bind to different metal sites on F1. The possible role of the metals in regulation of catalysis, and their relation to nucleotide binding is discussed.     

Cardiolipin mediates curcumin interactions with mitochondrial membranes

Curcumin, the main molecular ingredient of the turmeric spice, has been reported to exhibit therapeutic properties for varied diseases and pathological conditions. While curcumin appears to trigger multiple signaling pathways, the precise mechanisms accounting for its therapeutic activity have not been deciphered. Here we show that curcumin exhibits significant interactions with cardiolipin (CL), a lipid exclusively residing in the mitochondrial membrane. Specifically, we found that curcumin affected the structures and dynamics of CL-containing biomimetic and biological mitochondrial membranes. Application of several biophysical techniques reveals the CL-promoted association and internalization of curcumin into lipid bilayers. In parallel, curcumin association with CL containing bilayers increased their fluidity and reduced lipid ordering. These findings suggest that membrane modifications mediated by CL interactions may play a role in the therapeutic functions of curcumin, and that the inner mitochondrial membrane in general might constitute a potential drug target.     

Aflatoxin interactions with rat hepatic mitochondrial dehydrogenases

The effects of sublethal and possibly carcinogenic concentrations of aflatoxins B₁, B₂, G₁, G₂, and M₁ (AFB₁, AFG₂, AFG₁, AFG₂, and AFM₁) on rat hepatic mitochondrial dehydrogenases were studied by redox-dye titration. All the aflatoxins (minus AFG₂) enhanced the activities of the matrix dehydrogenases but had no marked effects on the membrane-bound enzymes. However, mitochondrial preincubation with each toxin raised the activities of all the dehydrogenases. AFG₂ was unique by inhibiting all the enzymes irreversibly.     

Phytoplasmas and their interactions with hosts

Phytoplasmas are bacteria without cell walls and are responsible for plant diseases that have large economic impacts. Knowledge of their biology is limited because they are uncultivable and experimentally inaccessible in their hosts. It is a mystery how these bacteria use the sugar-rich phloem sap in which they live and how they interact with the host. This makes it difficult to develop means to control them. Recently, the full genomes of two phytoplasmas have been sequenced, allowing new insights into their requirements. Phytoplasmas contain a minimal genome and lack genes coding for ATP synthases and sugar uptake and use, making them dependent on their host. This dependency can be exploited to elucidate the particular physiology of the phloem.     

Oncogenes and the mammalian X chromosome

Summary None of the up to now localized and expressed oncogenes maps to the mammalian X chromosome. This fact is discussed in the light of a trans-acting regulation mechanism for oncogenes. Such a specific regulation mechanism is demonstrated here for a qualitative change — i.e., varying timing of DNA-replication — at the putative c-myc gene locus in band 15E of murine T-cell leukemia. In intraspecific hybrids between tumor and non-tumor cells this qualitative change spreads over to all chromosomes 15 of the same cell, irrespective of their origin. This effects is thought to reflect a binary trans-acting regulation mechanism between homologous chromosomal loci. In the past specific chromosomal aberrations have been described in various tumors but none of these aberrations involve the X chromosome. For the mammalian X chromosome where there is usually only one gene copy per cell active the described kind of binary trans-acting regulation between homologous gene loci is rendered impossible.