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41.
42.
Lola Margulies 《Molecular & general genetics : MGG》1990,220(3):448-455
Summary An unusually high level of P-M hybrid dysgenesis in Drosophila melanogaster is characteristic of hybrid offspring originating from both, A (M × ) and B (P × M) crosses of a subline of the Harwich P strain, termed H
s
. The novel properties induced by mobility of P elements carried by H
s
paternal chromosomes include: very high (over 95%) gonadal dysgenesis (GD) in both sexes at the low restrictive temperature of 21°C, and highly premature sterility when males are reared at 18°C and aged at 21°C. Although all three major chromosomes of the H
s
subline contributed to this atypical pattern of gonadal dysgenesis, chromosome 3 had the largest effect. Gonadal dysgenesis showed a temperature- and sex-dependent repression pattern by the defective P elements of Muller-5 Birmingham chromosomes; at 21°C there was virtually no repression of male sterility, but most effective repression of GD in females. At 29°C repression was effective in males, but declined in females. The high thermosensitive sterility, low fecundity, and premature aging of the male germ line were greatly exacerbated when males derived from either A or B crosses were deficient either in excision repair (mei-9 mutation) or in post-replication repair (mei-41 mutation). These findings demonstrate that both DNA repair pathways are essential for the repair of lesions induced by P element transposition and support the hypothesis that P element-induced chromosome breaks are responsible for the virtual abolition of the germ line. The relatively high premature sterility of cross B DNA repair-deficient males, reared at 18°C and aged at 21°C, indicates that there is incomplete cytotype regulation in H
s
subline hybrids. 相似文献
43.
Mila Jankovic Tatjana Kostic Dragutin J. Savic 《Molecular & general genetics : MGG》1990,223(3):481-486
Summary Spontaneously arising histidine mutations in an Escherichia coli K12 strain deficient for DNA polymerase I were analysed at the DNA sequence level. We screened approximately 150000 colonies and isolated 106 histidine auxotrophs. Of these, 98 were unstable hisC mutations; 12 representative mutants analysed were shown to have arisen by the excision of a single quadruplet repeat in the sequence 5-GCTGGCTGGCTGGCTG-3. Of the eight mutations at other sites, three hisA deletions and one hisD deletion occurred as a consequence of misalignment of tandemly repeated pentamers (hisD) or decamers (hisA). A single hisA point mutation was found to be a missense mutation. Two extended deletions, covering the his operon were not analysed. We could not identify the hisC deletion by sequencing. We conclude that polA1 is a strong imitator that induces mutations mostly of the minus frameshift and deletion type by a Streisinger-type of mispairing in repetitive DNA sequences. Finally, the possible role of a 5-GTGG-3 sequence and its inverted or direct complements, which are found in the vicinity of all the deletions and frameshifts, is discussed. 相似文献
44.
Vanillin and its isomer o-vanillin have an effect on the adaptive and SOS responses, as well as mutagenesis, induced in Escherichia coli by N-methyl-N-nitrosourea (MNU) and UV irradiation, potentiating in some cases and suppressing in others. o-Vanillin markedly inhibited the MNU-induced adaptive response, while both vanillins potentiated the UV-induced SOS response. These phenomena appear to be responsible for the comutagenic or antimutagenic role of these chemicals in MNU and UV mutagenesis. 相似文献
45.
P. Pärt C. M. Wood 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1996,166(1):37-45
Using primary cultures of gill pavement cells from freshwater rainbow trout, a method is described for achieving confluent monolayers of the cells on glass coverslips. A continuous record of intracellular pH was obtained by loading the cells with the pH-sensitive flourescent dye 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and mounting the coverslips in the flowthrough cuvette of a spectrofluorimeter. Experiments were performed in HEPES-buffered media nominally free of HCO3. Resting intracellular pH (7.43 at extracellular pH=7.70) was insensitive to the removal of Cl– or the application of 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (0.1 mmol·l–1), but fell by about 0.3 units when Na+ was removed or in the presence of amiloride (0.2 mmol·l–1). Exposure to elevated ammonia (ammonia prepulse; 30 mmol·l–1 as NH4Cl for 6–9 min) produced an increase in intracellular pH (to about 8.1) followed by a slow decay, and washout of the pulse caused intracellular pH to fall to about 6.5. Intracellular non-HCO
3
–
buffer capacity was about 13.4 slykes. Rapid recovery of intracellular pH from intracellular acidosis induced by ammonia prepulse was inhibited more than 80% in Na+-free conditions or in the presence of amiloride (0.2 mmol·l–1). Neither bafilomycin A1 (3 mol·l–1) nor Cl removal altered the intracellular pH recovery rate. The K
m for Na+ of the intracellular pH recovery mechanism was 8.3 mmol·l–1, and the rate constant at V
max was 0.008·s–1 (equivalent to 5.60 mmol H+·l–1 cell water·min–1), which was achieved at external Na+ levels from 25 to 140 mmol·l–1. We conclude that intracellular pH in cultured gill pavement cells in HEPES-buffered, HCO
3
–
-free media, both at rest and during acidosis, is regulated by a Na+/H+ antiport and not by anion-dependent mechanisms or a vacuolar H+-ATPase.Abbreviations
BCECF
2,7-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein
-
BCECF/AM
2,7-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein, acetoxymethylester
-
Cholin-Cl
choline chloride
-
DMSO
dimethyl sulfoxide
-
EDTA
ethylene diamine tetra-acetic acid
-
FBS
foetal bovine serum
-
H
+
-ATPase
Proton-dependent adenosine triphosphatase
-
HEPES
N-[2-hydroxyethyl]piperazine-N[2-ethanesulfonic acid]
-
pH
i
intracellular pH
-
pH
e
extracellular pH
-
PBS
phosphate-buffered saline
-
SITS
4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid 相似文献
46.
Jonathan A. Lindquist Elisabeth Barofsky Philip N. McFadden 《Journal of Protein Chemistry》1996,15(1):115-122
Protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM, E.C. 2.1.1.77) was previously shown to be enzymatically methyl esterified in an autocatalytic manner at altered aspartyl residues; methyl esters are observed in a subpopulation of the enzyme termed thePCM fraction [Lindquist and McFadden (1994),J. Protein Chem.
13, 23–30]. The altered aspartyl sites serving as methyl acceptors inPCM have now been localized by using proteolytic enzymes and chemical cleavage techniques in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify fragments of the [3H]automethylated enzyme that contain a [3H]methyl ester. Methylation was positively identified at positions Asn188 and Asp217 in the enzyme sequence, a consequence of the spontaneous alteration of these sites tol-isoaspartyl ord-aspartyl sites and their methylation by active PCM molecules. The identification of more than one site of automethylation shows thatPCM is not a homogeneous population of damaged PCM molecules, but rather a complex population of molecules with a variety of age-altered damage sites.Abbreviations PCM
protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase
- EDTA
disodium ethylenediaminetetraacetate
- PMSF
phenylmethylsulfonyl fluoride
- TEA
trifluoroacetic acid
- HPLC
high-pressure liquid chromatography 相似文献
47.
Protein-L-isoaspartate (D-aspartate) O-methyltransferases (EC 2.1.1.77) that catalyze the transfer of methyl groups from S-adenosylmethionine to abnormal L-isoaspartyl and D-aspartyl residues in a variety of peptides and proteins are widely distributed in procaryotes and eucaryotes. These enzymes participate in the repair of spontaneous protein damage by facilitating the conversion of L-isoaspartyl and D-aspartyl residues to normal L-aspartyl residues. In this work, we have identified an L-isoaspartyl methyltransferase activity in Arabidopsis thaliana, a dicotyledonous plant of the mustard family. The highest levels of activity were detected in seeds. Using degenerate oligonucleotides corresponding to two highly conserved amino acid regions shared among the Escherichia coli, wheat, and human enzymes, we isolated and sequenced a full-length genomic clone encoding the A. thaliana methyltransferase. Several methyltransferase cDNAs were also characterized, including ones that would encode full-length polypeptides of 230 amino acid residues. Messenger RNAs for the A. thaliana enzyme were found in a variety of tissues that did not contain significant amounts of active enzyme suggesting the possibility of translational or posttranslational controls on methyltransferase levels. We have identified a putative abscisic acid-response element (ABRE) in the 5-untranslated region of the A. thaliana L-isoaspartyl methyltransferase gene and have shown that the expression of the mRNA is responsive to exogenous abscisic acid (ABA), but not to the environmental stresses of salt or drought. The expression of the A. thaliana enzyme appears to be regulated in a distinct fashion from that seen in wheat or in animal tissues. 相似文献
48.
To study the mechanism of protein carboxyl methyltransferase-driven repair of age-damaged sites in polypeptides, a modell-isoaspartyl peptide,l-isotetragastrin, was enzymatically repaired to normall-tetragastrin in the presence of18O-enriched water. By this design, the enrichment of18O atoms in the peptide would reflect the number of passages through a hydrolyzable succinimide intermediate during formation of the repaired product. Mass determinations by FAB mass spectrometry revealed repaired peptide with two18O atoms incorporated, demonstrating that more than a single cycle of methylation and demethylation is necessary to ensure stoichiometric repair.Abbreviations HPLC
high-pressure liquid chromatography
- FAB
fast atom bombardment
- TFA
trifluoroacetic acid
- PCM
proteind-aspartyl/L-isoaspartyl carboxyl methyltransfer-ase
-
l-Normal
[l-Asp3]tetragastrin
-
l-Iso
[L-isoAsp3]tetragastrin
-
d-Normal
[d-Asp3]tetragastrin
-
d-Iso
[d-isoAsp3]tetragastrin 相似文献
49.
A question that is central to understanding the mechanisms of aging and cellular deterioration is whether enzymes involved in recognition and metabolism of spontaneously damaged proteins are themselves damaged, either becoming substrates for their own activity; or being unable to act upon themselves, initiating cascades of cellular damage. We show here byin vitro experiments that protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM) from bovine erythrocytes does methylate age-dependent amino acid damage in its own sequence. The subpopulation that is methylated, termed thePCM fraction, appears to be formed through age-dependent deamidation of an asparaginyl site to either anl-isoaspartyl ord-aspartyl site because (a) the stoichiometry of automethylation of purified PCM is less than 1%, a value typical of the substoichiometric methylation of many other aged protein substrates, (b)PCM is slightly more acidic than the bulk of PCM, and (c) the methyl esterified site inPCM has the characteristic base-lability of this type of methyl ester. Also, the methyl group is not incorporated into the enzyme as an active site intermediate because the incorporated methyl group is not chased onto substrate protein. The effect of enzyme dilution on the rate of the automethylation reaction is consistent with methylation occurring between protein molecules, showing that the pool of PCM is autocatalytic even though individual molecules may not be. The automethylation and possible self-repair of the PCM pool has implications for maintaining thein vivo efficiency of methylation-dependent protein repair. 相似文献
50.
The acinous salivary glands of the cockroach (Periplaneta americana) consist of four morphologically different cell types with different functions: the peripheral cells are thought to produce the fluid component of the primary saliva, the central cells secrete the proteinaceous components, the inner acinar duct cells stabilize the acini and secrete a cuticular, intima, whereas the distal duct cells modify the primary saliva via the transport of water and electrolytes. Because there is no direct information available on the distribution of ion transporting enzymes in the salivary glands, we have mapped the distribution of two key transport enzymes, the Na+/K+-ATPase (sodium pump) and a vacuolar-type H+-ATPase, by immunocytochemical techniques. In the peripheral cells, the Na+/K+-ATPase is localized to the highly infolded apical membrane surface. The distal duct cells show large numbers of sodium pumps localized to the basolateral part of their plasma membrane, whereas their highly folded apical membranes have a vacuolar-type H+-ATPase. Our immunocytochemical data are supported by conventional electron microscopy, which shows electrondense 10-nm particles (portasomes) on the cytoplasmic surface of the infoldings of the apical membranes of the distal duct cells. The apically localized Na+/K+-ATPase in the peripheral cells is probably directly involved in the formation of the Na+-rich primary saliva. The latter is modified by the distal duct cells by transport mechanisms energized by the proton motive force of the apically localized V-H+-ATPase. 相似文献