Vitronectin exists in two structurally and functionally distinct forms in human plasma

Vitronectin (serum spreading factor, S-protein or epibolin) is a plasma glycoprotein implicated in cell adhesion, as well as in the regulation of complement-mediated cytolysis and antithrombin III function. Vitronectin was found to exist in fresh human plasma as a heterogeneous mixture consisting of 2% heparin-binding form and the remainder as a non-binding species. Heparin-binding vitronectin consisted of 6.5 S aggregates with a Stokes radius of 5.6 nm, which was enriched in the 65 kDa polypeptide, with a high content of molecules and a putative unfolded conformation. In contrast, non-heparin-binding vitronectin was a 4.2 S monomer with a Stokes radius of 3.9 nm, which appeared to be in a folded conformation with an immunologically cryptic site. Both vitronectins displayed similar activities in mediating the spreading of BHK fibroblastic cells on substrates. During blood coagulation, 5% more of the non-heparin-binding vitronectin was converted into the heparin-binding form, producing a greater than 3.5-fold increase in this species. Our results indicate that vitronectin normally exists in circulating blood in at least two structurally and functionally distinct forms which may serve different functions.     

Two distinct DNA ligases from Drosophila melanogaster embryos

Embryos of Drosophila melanogaster contain two distinct DNA ligases (DNA ligase I and II). DNA ligase I was eluted at 0.2 M KCl and DNA ligase II at 0.6 M KCl on phosphocellulose column chromatography. The former was rich in early developing embryos and its activity decreased during embryonic development. The latter was found constantly throughout the developing stages of embryos. DNA ligase I existed in a cytoplasmic fraction and DNA ligase II is concentrated in nuclei. Both enzymes ligate 5'-phosphoryl and 3'-hydroxyl groups in oligo(dT) in the presence of poly(dA). DNA ligase II is also able to join oligo(dT)(poly(rA). Both enzymes require ATP and Mg2+ for activity. The Km for ATP is 2.7 X 10(-6) M for DNA ligase I, and 3.0 X 10(-5) M for DNA ligase II. DNA ligase I requires dithiothreitol and polyvinyl alcohol, but DNA ligase II does not. Both enzymes are inhibited in the presence of N-ethylmaleimide. DNA ligase I is active at a low salt concentration (0-30 mM KCl), but DNA ligase II is active at high salt concentrations (50-100 mM). DNA ligase I is more labile than DNA ligase II. The molecular masses of DNA ligase-AMP adducts were determined as 86 and 75 kDa for DNA ligase I, and as 70 (major protein) and 90 kDa (minor protein) for DNA ligase II under denaturing conditions. A sedimentation coefficient of 4.2 S was observed for DNA ligase II. Consequently, Drosophila DNA ligase I and II are quite similar to mammalian DNA ligase I and II. Drosophila DNA ligase I and a DNA ligase by B.A. Rabin et al. [(1986) J. Biol. Chem. 261, 10637-10645] seem to be the same enzyme.     

Intramolecular heterogeneity of mitochondrial DNA of Drosophila melanogaster

DNA from purified mitochondria of Drosophila melanogaster can be isolated as supercoiled molecules which when nicked have a contour length of 5.9 micron. Partial denaturation mapping shows regional heterogeneity of base composition with one early denaturing region, with a calculated GC content close to zero, extending over 20% of the genome. DNA isolated from unfertilized eggs shows nuclear and mitochondrial DNA in equal proportions; we found no evidence of other cytoplasmic species.     

Accumulation of deleted mitochondrial DNA in aging Drosophila melanogaster

Cumulative damage in mitochondria by reactive oxygen species is thought to result in a decrease in mitochondrial respiratory function and to contribute to the age-related decline in the physiological function of organisms. The mitochondrial genome is also subjected to damage with age through deletions. The accumulation of deleted mitochondrial DNA (mtDNA) has been observed in various animals, but still remains unclear in insects. We examined the accumulation of deleted mtDNA in D. melanogaster at various ages from larvae to 65-day-old adults. When DNA extracted from whole bodies was examined by PCR and Southern hybridization, the age-related accumulation of deletions was not clear. However, when the accumulation of deleted mtDNA with age was examined separately in three parts of the body (head, thorax and abdomen), deleted mtDNA signals were detected more frequently in the thorax and the accumulation was age-dependent. Three of the deleted mtDNA were cloned, and the breakpoints of the deletions were identified. These results strongly suggest that deleted mtDNA accumulates in Drosophila with age in a tissue-specific manner.     

Mitochondrial DNA variants influence mitochondrial bioenergetics in Drosophila melanogaster

The influence of mitochondrial DNA (mtDNA) mutations on human disease has been extensively studied, but the impact of mutations within the adaptive range is debated. We studied males from lines of Drosophila melanogaster that have a highly standardized nuclear genome but different mtDNA, at two ages. We measured mitochondrial respiration on permeabilized muscle fibers, hydrogen peroxide production of isolated mitochondria and mtDNA copy number of whole individuals. The results show that a small set of naturally occurring mtDNA mutations can have a significant influence on mitochondrial bioenergetics that may change as the organism ages.     

Three forms of DNA polymerase from Drosophila melanogaster embryos. Purification and properties.

DNA polymerase was purified from Drosophila melanogaster embryos by a combination of phosphocellulose adsorption, Sepharose 6B gel filtration, and DEAE-cellulose chromatography. Three enzyme forms, designated enzymes I, II, and III, were separated by differential elution from DEAE-cellulose and were further purified by glycerol gradient centrifugation. Purification was monitored with two synthetic primer-templates, poly(dA) . (dT)-16 and poly(rA) . (dT)-16. At the final step of purification, enzymes I, II, and III were purified approximately 1700-fold, 2000-fold and 1000-fold, respectively, on the basis of their activities with poly(dA) . (dT)-16. The DNA polymerase eluted heterogeneously as anomalously high-molecular-weight molecules from Sepharose 6B gel filtration columns. On DEAE-cellulose chromatography enzymes I and II eluted as distinct peaks and enzyme III eluted heterogeneously. On glycerol velocity gradients enzyme I sedimented at 5.5-7.3 S, enzyme II sedimented at 7.3-8.3 S, and enzyme III sedimented at 7.3-9.0 S. All enzymes were active with both synthetic primer-templates, except the 9.0 S component of enzyme III, which was inactive with poly(rA) . (dT)-16. Non-denaturing polyacrylamide gel electrophoresis did not separate poly(dA) . (dT)-16 activity from poly(rA) . (dT)-16 activity. The DNA polymerase preferred poly(dA) . (dT)-16 (with Mg2+) as a primer-template, although it was also active with poly(rA) . (dT)-16 (with Mn2+), and it preferred activated calf thymus DNA to native or heat-denatured calf thymus DNA. All three primer-template activities were inhibited by N-ethylmaleimide. Enzyme activity with activated DNA and poly(dA) . (dT)-16 was inhibited by K+ and activity with poly(rA) . (dT)-16 was stimulated by K+ and by spermidine. The optimum pH for enzyme activity with the synthetic primer-templates was 8.5. The DNA polymerases did not exhibit deoxyribonuclease or ATPase activities. The results of this study suggest that the forms of DNA polymerase from Drosophila embryos have physical properties similar to those of DNA polymerase-alpha and enzymatic properties similar to those of all three vertebrate DNA polymerases.     

Closely related DNA sequences specify distinct patterns of developmental expression in Drosophila melanogaster.

Three short synthetic DNA sequences, which are closely related to one another, confer three distinct patterns of developmental expression on the heat shock hsp70 gene in transgenic Drosophila melanogaster lines. These results show that small variations or even single base pair changes in a repeated element of a regulatory sequence can create promoters that display new specificities of tissue and developmental regulation. Interestingly, the three patterns of developmental expression conferred by the synthetic DNAs resemble in part those of the known developmental genes: glucose dehydrogenase (Gld), Dopa decarboxylase (Ddc), and salivary gland secretory proteins (Sgs), respectively. In each case, the defined regulatory region of the known developmental gene contains multiple sequences that are similar or identical to the synthetic sequence that confers a similar pattern of developmental expression on the hsp70 gene. Thus, these results are congruent with the view that short sequence elements in multiple copies can confer either simple or relatively complex patterns of developmental expression on a receptive promoter like that of hsp70. Furthermore, the fact that the three variants tested produced three distinct patterns of expression in transgenic animals suggests that the number of different elements is large.     

Direct estimation of the mitochondrial DNA mutation rate in Drosophila melanogaster

Mitochondrial DNA (mtDNA) variants are widely used in evolutionary genetics as markers for population history and to estimate divergence times among taxa. Inferences of species history are generally based on phylogenetic comparisons, which assume that molecular evolution is clock-like. Between-species comparisons have also been used to estimate the mutation rate, using sites that are thought to evolve neutrally. We directly estimated the mtDNA mutation rate by scanning the mitochondrial genome of Drosophila melanogaster lines that had undergone approximately 200 generations of spontaneous mutation accumulation (MA). We detected a total of 28 point mutations and eight insertion-deletion (indel) mutations, yielding an estimate for the single-nucleotide mutation rate of 6.2 × 10⁻⁸ per site per fly generation. Most mutations were heteroplasmic within a line, and their frequency distribution suggests that the effective number of mitochondrial genomes transmitted per female per generation is about 30. We observed repeated occurrences of some indel mutations, suggesting that indel mutational hotspots are common. Among the point mutations, there is a large excess of G→A mutations on the major strand (the sense strand for the majority of mitochondrial genes). These mutations tend to occur at nonsynonymous sites of protein-coding genes, and they are expected to be deleterious, so do not become fixed between species. The overall mtDNA mutation rate per base pair per fly generation in Drosophila is estimated to be about 10× higher than the nuclear mutation rate, but the mitochondrial major strand G→A mutation rate is about 70× higher than the nuclear rate. Silent sites are substantially more strongly biased towards A and T than nonsynonymous sites, consistent with the extreme mutation bias towards A+T. Strand-asymmetric mutation bias, coupled with selection to maintain specific nonsynonymous bases, therefore provides an explanation for the extreme base composition of the mitochondrial genome of Drosophila.     

RGS7 is palmitoylated and exists as biochemically distinct forms

Regulator of G protein signaling (RGS) proteins are GTPase-activating proteins that modulate neurotransmitter and G protein signaling. RGS7 and its binding partners Galpha and Gbeta5 are enriched in brain, but biochemical mechanisms governing RGS7/Galpha/Gbeta5 interactions and membrane association are poorly defined. We report that RGS7 exists as one cytosolic and three biochemically distinct membrane-bound fractions (salt-extractable, detergent-extractable, and detergent-insensitive) in brain. To define factors that determine RGS7 membrane attachment, we examined the biochemical properties of recombinant RGS7 and Gbeta5 synthesized in Spodoptera frugiperda insect cells. We have found that membrane-bound but not cytosolic RGS7 is covalently modified by the fatty acid palmitate. Gbeta5 is not palmitoylated. Both unmodified (cytosolic) and palmitoylated (membrane-derived) forms of RGS7, when complexed with Gbeta5, are equally effective stimulators of Galpha(o) GTPase activity, suggesting that palmitoylation does not prevent RGS7/Galpha(o) interactions. The isolated core RGS domain of RGS7 selectively binds activated Galpha(i/o) in brain extracts and is an effective stimulator of both Galpha(o) and Galpha(i1) GTPase activities in vitro. In contrast, the RGS7/Gbeta5 complex selectively interacts with Galpha(o) only, suggesting that features outside the RGS domain and/or Gbeta5 association dictate RGS7-Galpha interactions. These findings define previously unrecognized biochemical properties of RGS7, including the first demonstration that RGS7 is palmitoylated.     

The characterization of multiple forms of kynurenine formidase in Drosophila melanogaster.

Two enzymic forms of kynurenine formamidase (EC 3.5.1.9) from Drosophila melanogaster were separated and partially purified by pH fractionation, (NH4) 2SO4 fractionation and Sephadex G-75 gel filtration. The enzymes were also separated by DEAE-cellulose ion-exchange chromatography and distinguished by their different rates of thermal inactivation. The multiple forms are termed formamidase I and formamidase II. The molecular weight of formamidase I as measured by Sephadex G-75 chromatography is 60 000 and that of formamidase II is 31 000. The pH optima are broad, ranging between 6.7 and 7.8 for formamidase I and 6.5 and 8.0 for formamidase II. The apparent Km values are 5-10(-3) and 0.83-10(-3) M, resepctively. The possibility that formamidase II is an active subunit of formamidase I is discussed, although neither enzyme will convert to the other when separated and rechromatographed. Eight organisms were tested for the presence or absence of multiple forms of formamidase. Drosophila melanogaster and Drosophila virilis have both enzymes; cow, chicken, yeast and housefly have formamidase I only, and mouse and frog have formamidase II only.     

The distribution of two highly repeated DNA sequences within Drosophila melanogaster chromosomes

In situ hybridization using ³H-RNA probes has been used to localize the sequences found in two satellites of density 1.705 g/cc and 1.672 g/ cc to specific sites within the chromosomal complement. A detailed analysis of the sites on the X chromosome was carried out using the scute series of inversions to relate the heterochromatic breakpoint relative to the location of the sequence on this chromosome. It has also been possible to establish the order of arrangement of 1.705 and 1.672 DNA at the heterochromaticeuchromatic junction on chromosome 3(R). A mitotic map is provided. The T_m of hybrids formed in situ showed that the hybrids were representative of the sequences being analyzed. The two satellites also were traced through a number of purification procedures to show that a covalent linkage may be likely between the 1.705 g/cc and 1.672 g/cc satellite as predicted from in situ hybridization analyses.     

Molecular cloning of part of the mitochondrial DNA of Drosophila melanogaster

We report the construction of recombinant plasmids containing part of the mitochondrial DNA of $\text{Drosophila}\text{melanogaster}$. Of the four fragments of this DNA generated by the restriction endonuclease HindIII, two were successfully cloned into the HindIII site of the plasmid pCM2. Unexpectedly the other two fragments could not be isolated by cloning into the HindIII site of either pCM2 or pBR322. Part of a third fragment, containing the gene for the large ribosomal RNA, was incorporated into the PstI site of pBR322. We show that this recombinant plasmid contains sequences complementary to an abundant RNA species which is present in $\text{Drosophila}$ embryos and which binds to oligo-dT-cellulose.     

Two distinct domains in Drosophila melanogaster telomeres

Telomeres are generally considered heterochromatic. On the basis of DNA composition, the telomeric region of Drosophila melanogaster contains two distinct subdomains: a subtelomeric region of repetitive DNA, termed TAS, and a terminal array of retrotransposons, which perform the elongation function instead of telomerase. We have identified several P-element insertions into this retrotransposon array and compared expression levels of transgenes with similar integrations into TAS and euchromatic regions. In contrast to insertions in TAS, which are silenced, reporter genes in the terminal HeT-A, TAHRE, or TART retroelements did not exhibit repressed expression in comparison with the same transgene construct in euchromatin. These data, in combination with cytological studies, provide evidence that the subtelomeric TAS region exhibits features resembling heterochromatin, while the terminal retrotransposon array exhibits euchromatic characteristics.     

Further study on selective transmission of mitochondrial DNA in heteroplasmic lines of Drosophila melanogaster.

The temperature-dependent transmission of mitochondrial DNA (mtDNA) was investigated in heteroplasmic lines of Drosophila melanogaster established by germ-plasm transplantation. Using D. melanogaster, D. simulans and D. mauritiana as germ-plasm donors, five recipient-donor combinations of heteroplasmy, differing from those previously examined (Matsuura et al., 1991), were constructed. For intraspecific reciprocal combinations, donor mtDNA in one combination was retained at 25 degrees C but was almost lost by the tenth generation at 19 degrees C. In the reciprocal, the proportion of the same type of recipient mtDNA decreased more quickly at 19 degrees C than 25 degrees C. Decreasing rates at 19 degrees C in the reciprocals differed from each other. For interspecific combinations, two species were used as germ-plasm donors. Donor mtDNA derived from D. simulans was lost at both temperatures and the rate of decrease was greater at 19 degrees C than 25 degrees C. The proportion of donor mtDNA derived from D. mauritiana increased at a greater rate at 25 degrees C than 19 degrees C when using two different strains of D. melanogaster as recipients. These results suggest that both the nuclear and two types of mitochondrial genomes are involved in the selective transmission of mtDNA.     

The B-A transition in superhelical DNA.

Relaxation of a DNA superhelical stress due to the B to A transition induced by trifluoroethanol has been studied by assessing the change of DNA orientation in a flow gradient. Using DNAs of different superhelical densities, a decrease in the winding angle during the B----A shift of DNA was found to be 1.5 degrees per base pair in solution. Accepting the winding angle for B-DNA in solution to be 34.1 degrees, that for A-DNA must have a value of 32.6 degrees which agrees with the X-ray data for A-DNA in the condensed state. The date obtained within the B-A transition interval make it possible to conclude that there is an increase in winding at each B/A junction, which is about 5 degrees per one junction.     

Fluctuations in superhelical DNA.

The effect of superhelicity on the base-pair opening probability and on the probability of occurrence of cruciform states in palindromic regions is theoretically treated. The calculations show that below the superhelix density value of -sigma=0.05 superhelicity does not appreciably affect the characteristics of DNA secondary structure fluctuations. In the range of physiological superhelix densities sigma (-sigma=0.05-0.09) the base-pair opening probability markedly increases. However, within this range of sigma the base-pairs are opened only transiently and permanently open regions are not formed. Permanently opened regions appear at higher negative superhelix densities (-sigma greater than 0.10). At the values of -sigma higher than 0.06 a cruciform structure in the palindromic region centred in position 3965 proves to be the most probable fluctuational disturbance in the 0x174 duplex DNA. Different experimental approaches used for probing the fluctuations in superhelical DNA have been analysed. The results suggest that most direct quantitative information can be derived from data on the nicking of closed DNA by single strand-specific endonucleases. Such data (Wang, 1974) accord with the results of theoretical calculations. Calculations show that, due to base-pair opening, the total free energy of superhelical DNA should depend parabolically on sigma only up to some critical value of sigma=sigmac. If negative superhelicity exceeds this critical value, which under physiological conditions proves to be -sigma=0.085, the free energy should increase linearly with -sigma. The biological role of supercoiling is discussed in the light of obtained results.