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1.
The 1.672 g/cm3 satellite DNA of Drosophila melanogaster was purified by successive equilibrium centrifugations in a CsCl gradient, an actinomycin DCsCl gradient, and a netropsin sulfate/CsCl gradient. The resulting DNA was homogeneous by the physical criteria of thermal denaturation, renaturation kinetics and equilibrium banding in each of the gradients listed above. In addition, the complementary strands could be separated in an alkaline CsCl gradient. Despite this rigorous purification procedure, nucleotide sequence analysis indicates the presence of two different DNA species in this satellite, poly A-A-T-A-TT-T-A-T-A and polyA-A-T-A-T-A-TT-T-A-T-A-T-A. Further physical, chemical and template properties of the isolated complementary strands demonstrate that these two repeating sequences are not interspersed with each other. This result has biological significance since sequences of this particular satellite are known to be located primarily on two different chromosomes, Y and 2. These results further suggest that the sequence heterogeneity observed in satellite DNA of higher eukaryotes may result from mixtures of very closely related but molecularly homogeneous repeated sequences each restricted to a particular chromosome or chromosomal region.  相似文献   

2.
Highly repeated satellite DNAs often consist of mixtures of DNAs with closely related repeating sequences. By cloning individual molecules we have resolved the 1.705 g/cm3 satellite DNA of Drosophila melanogaster into two distinct components: polydA-A-G-A-GT-T-C-T-C and polydA-A-G-A-G-A-GT-T-C-T-C-T-C. The presence of two distinct sequences within this physically homogeneous satellite DNA had not previously been detected by standard physical, chemical, or sequencing techniques. Both cloning and direct sequence analysis suggest that the five-base-pair and seven-base-pair repeating units reside on separate molecules and are not interspersed with each other.  相似文献   

3.
An analysis of the repeat structure of the highly repetitive sequence, component α DNA of the African green monkey, shows that the DNA contains restriction sites for EcoRI, EcoRI1, HindIII and HaeIII. All four restriction enzyme activities indicate a basic repeat length of 176 ± 4 base-pairs. In addition to primary EcoRI1 and HindIII sites, about 59% of the repeat sequences contain secondary EcoRI1 sites and about 36% of the repeat sequences contain secondary HindIII sites. The secondary sites are located less than 176 base-pairs from the primary sites and their cleavage yields several complex series of minor, intermediate segments in gels of the partial EcoRI1 or HindIII digests. Cleavage at the secondary sites yields segments shorter than the unit monomer in the limit digests. The sites for EcoRI, EcoRI1, HindIII and HaeIII have been mapped within the repeat unit.Treatment of the monkey nuclei with micrococcal nuclease at 2 °C and in the presence of 80 mm-NaCl reveals two distinct populations of nucleosomes. One population contains bulk DNA sequences, and after cleavage with micrococcal nuclease this population yields heterogeneous segments of DNA spanning 180 to 200 base-pairs in length. The other population contains component α sequences and after cleavage with micrococcal nuclease yields homogeneous segments of component α DNA that are exact multiples of the basic sequence repeat unit of 176 base-pairs. Thus, the cleavage by micrococcal nuclease of nucleosomal arrays containing component α sequences is as regular and precise as the cleavage of the purified DNA by the restriction enzymes. The resolution of the two distinct subsets of nucleosomes in the monkey nuclei is dependent upon the conditions of ionic strength and temperature employed during the nuclear isolation and the micrococcal nuclease digestion.These observations are consistent with a phase relation between the component α repeat sequences and the associated nucleosomal proteins (Musich et al., 1977b). They are also in accord with the hypothesis that the subunit structure of constitutive heterochromatin modulates or determines the repeat sequence structure and hence, the evolution of many highly repetitive mammalian DNAs (Maio et al., 1977).  相似文献   

4.
The DNA sequence recognised by the HinfIII restriction endonuclease   总被引:3,自引:0,他引:3  
HinfIII is a type III restriction enzyme (Kauc &; Piekarowicz, 1978) isolated from Haemophilus influenzae Rf. Like other type III restriction endonucleases, the enzyme also catalyses the modification of susceptible DNA. It requires ATP for DNA cleavage and S-adenosyl methionine for DNA methylation. We have determined the DNA sequence recognised by HinfIII to be:
5′-C-G-A-A-T-3′·····3′-G-C-T-T-A-5′
In restriction, the enzyme cleaves the DNA about 25 base-pairs to the right of this sequence. In the modification reaction only one of the strands is methylated, that containing the 5′-C-G-A-A-T-3′ sequence.  相似文献   

5.
6.
We have determined the complete nucleotide sequence of the monomer repeating unit of the 1.688 g/cm3 satellite DNA from Drosophila melanogaster. This satellite DNA, which makes up 4% of the Drosophila genome and is located primarily on the sex chromosomes, has a repeat unit 359 base-pairs in length. This complex sequence is unrelated to the other three major satellite DNAs present in this species, each of which contains a very short repeated sequence only 5 to 10 base-pairs long. The repeated sequence is more similar to the complex repeating units found in satellites of mammalian origin in that it contains runs of adenylate and thymidylate residues. We have determined the nature of the sequence variations in this DNA by restriction nuclease cleavage and by direct sequence determination of (1) individual monomer units cloned in hybrid plasmids, (2) mixtures of adjacent monomers from a cloned segment of this satellite DNA, (3) mixtures of monomer units isolated by restriction nuclease cleavage of total 1.688 g/cm3 satellite DNA. Both direct sequence determination and restriction nuclease cleavage indicate that certain positions in the repeat can be highly variable with up to 50% of certain restriction sites having altered recognition sequences. Despite the high degree of variation at certain sites, most positions in the sequence are highly conserved. Sequence analysis of a mixture of 15 adjacent monomer units detected only nine variable positions out of 359 base-pairs. Total satellite DNA showed only four additional positions. While some variability would have been missed due to the sequencing methods used, we conclude that the variation from one repeat to the next is not random and that most of the satellite repeat is conserved. This conservation may reflect functional aspects of the repeated DNA, since we have shown earlier that part of this sequence serves as a binding site for a sequence-specific DNA binding protein isolated from Drosophila embryos (Hsieh &; Brutlag, 1979).  相似文献   

7.
The periodicities of the restriction enzyme cleavage sites in highly repetitive DNAs of six mammalian species (monkey, mouse, sheep, human, calf and rat) appear related to the length of DNA contained in the nucleosome subunit of chromatin. We suggest that the nucleosome structure is an essential element in the generation and evolution of repeated DNA sequences in mammals (Brown et al., 1978; Maio et al., 1977). The possibility of a phase relation between DNA repeat sequences and associated nucleosome proteins is consistent with this hypothesis and has been tested by restriction enzyme and micrococcal nuclease digestions of repetitive DNA sequences in isolated, intact nuclei.Sites for four different restriction enzyme activities, EcoRI, EcoRI1, HindIII and HaeIII have been mapped within the repeat unit of component α DNA, a highly repetitive DNA fraction of the African green monkey. The periodicity of cleavage sites for each of the enzymes (176 ± 4 nucleotide base-pairs) corresponds closely to the periodicity (about 185 nucleotide base-pairs) of the sites attacked in the initial stages of micrococcal nuclease digestion of nuclear chromatin. In intact monkey nuclei, EcoRI-RI1 sites are accessible to restriction enzyme cleavage; the HindIII and HaeIII sites are not. The results suggest (1) that, in component α chromatin, the EcoRI-RI1 sites are found at the interstices of adjacent nucleosomes and (2) the HindIII and HaeIII sites are protected from cleavage by their location on the protein core of the nucleosome. This interpretation was confirmed by experiments in which DNA segments of mononucleosomes and nucleosome cores released from CV-1 nuclei by micrococcal nuclease were subsequently treated with EcoRI, EcoRI1 and HindIII. A major secondary segment of component α, about 140 nucleotide base-pairs in length, was released only by treatment with HindIII, in keeping with the location of the HindIII sites in the restriction map and their resistance to cleavage in intact nuclei.EcoRI reduces calf satellite I DNA to a segment of about 1408 nucleotide basepairs. In contrast, restriction of calf satellite I DNA with EcoRI1 produces six prominent segments ranging in size from 176 to 1408 nucleotide base-pairs. Treatment of isolated calf nuclei with either EcoRI or EcoRI1 did not produce segments shorter than 1408 base-pairs, indicating that while canonical EcoRI sites are accessible to attack, the irregularly spaced EcoRI1 sites are specifically blocked. The results are consistent with a phase relation between the repeat sequence of calf satellite I DNA and an octameric array of nucleosomes.  相似文献   

8.
Cytochrome P1-450 (P1-450) is defined as that cytochrome most closely associated with 3-methylcholanthrene (MC)-induced aryl hydrocarbon hydroxylase (AHH) activity. Recently a cloned DNA sequence (clone 46) was shown to represent a portion of the P1-450 structural gene [Negishi etal., Proc. Nat. Acad. Sci. U.S.A.78: 800–804 (1981)]. Poly(A+)-enriched RNA was isolated from total liver homogenate, membrane-bound polysomes and from free polysomes at various times after MC treatment of Ah-responsive C57BL6N (B6) and Ah-nonresponsive DBA2N (D2) inbred mice. The poly(A+)-enriched RNA was separated by methylmercury-agarose gel electrophoresis and hybridized to nick-translated [32P]DNA from clone 46. By means of this RNA-DNA hybridization, only 6% of total polysomal P1-450 mRNA exists in free polysomes after 24 h of MC treatment. The data indicate that the endoplasmic reticulum is the principal site of synthesis for this integral microsomal protein.Studies of induction kinetics following MC treatment provided the evidence of the rapid increase of total liver and membrane bound P1-450 mRNA preceding the synthesis of apo-P1-450 and the increase of AHH activity.  相似文献   

9.
Detection of sequence heterology by use of the N. Crassa nucleases   总被引:1,自引:0,他引:1  
We have used the single-strand specific nucleases of Neurospora crassa to detect sequence divergencies between two similar DNA molecules: restriction endonuc lease EcoRI produced linears from Simian Virus 40 and a variant of human origin, DAR. Enzyme treatment of the heteroduplex DNA resulted in specific cleavage into two fragments of one-third and two-thirds genome length. These two viral DNAs therefore have at least one region of heterology located about 0.35 map units from the EcoRI site. Due to the known specificities of the N.crassa nucleases, this technique is applicable to detect mutations in RNA or DNA genomes.  相似文献   

10.
α-Satellite DNA from African green monkey cells was analysed with restriction nucleases in some detail confirming and complementing our earlier results. With EcoRI and HaeIII (or BsuRI isoschizomer), about 25 and 10%, respectively, of the satellite DNA were cleaved into a series of fragments of the 172 bp repeat length and multiples thereof. To allow studies with fragments of homogeneous sequence unit length, HindIII fragments were covalently joined with the plasmid pBR313. After transformation 19 clones were obtained, containing up to three monomer fragments. Nine of the clones were characterized by digestion with EcoRI. Three of these had cleavage sites for this nuclease in the satellite DNA portion. In the six clones tested with HaeIII no cleavage site was detected in the cloned DNA. The results are discussed in relation to the nucleotide sequence data recently published by Rosenberg et al. (1978) and in the context of random and nonrandom processes in satellite DNA evolution.  相似文献   

11.
The acid phosphatase isolated from sweet potato tubers by us is unique Mn(III)-containing enzyme which hydrolyzes phosphomonoesters and nucleotide phosphates. The present 31P and 17O NMR studies of the Mn(III)-containing acid phosphatase solved two important problems. The broadening of the phosphate 31P resonance signal in the 1:1 enzyme-substrate system shows evidence for direct metal-phosphate interaction in the Mn(III)-containing acid phosphatase. In addition, the 17O NMR evidence for oxygen exchange from water into inorganic phosphate strongly indicates that the Mn(III)-containing acid phosphatase catalyzes an apparent transition state displacement and P-O cleavage as follows: ROPO3= + H17OHROH + H17OPO3=.  相似文献   

12.
13.
Mitochondrial DNA from Drosophila contains high “A+T”-rich region. Its DNA replication starts in the “A+T”-rich region and proceeds unidirectionally around the molecule. In order to determine precise location of the DNA replication origin and elucidate unique feature of its nucleotide sequence, the “A+T”-rich region of mitochondrial DNA from Drosophilavirilis has been cloned in Escherichiacoli. The chimeric plasmid DNA containing the “A+T”-rich region stimulates invitro DNA replication system from Drosophilavirilis mitochondria about ten fold higher than the parental plasmid DNA, as does native mitochondrial DNA.  相似文献   

14.
Synthesis of T4 tRNAGln depends on normal levels of Escherichiacoli ribonuclease III. Infection of cell strains carrying a mutation in the gene for this enzyme resulted in severe depression in tRNAGln production, as revealed by chemical and suppressor tRNA analyses. The remaining seven T4 tRNAs were synthesized in the mutant cells. The requirement of ribonuclease III for synthesis of tRNAGln points to an essential cleavage by the enzyme of a precursor RNA containing tRNAGln.  相似文献   

15.
Long range periodicities in mouse satellite DNA.   总被引:66,自引:0,他引:66  
Escherichia coli restriction enzyme II breaks mouse satellite DNA into fragments which form a series of bands on gel electrophoresis. The DNA in the strongest band has a length of 220 to 260 nucleotide pairs and the other bands are multiples of this length. It is shown that these fragments are linked together in long arrays in the satellite sequence. The reassociation register of the DNA is about half the length of the 220 to 260 nucleotide pair fragment. In the electrophoresis pattern of the Eco RII2 fragments other weaker bands can be seen. The stronger bands of the minor patterns fall half-way between the bands of the main pattern and the smallest is 120 to 130 nucleotide pairs long. The properties of the minor fragments suggest short spacings of the restriction site which have been produced by unequal crossing-over. The extents of divergence and unequal crossing-over are estimated. From this analysis and the sequence analysis described in the accompanying paper (Biro et al., 1975) it is proposed that mouse satellite DNA consists of an hierarchy of four periodicities which reflect stages in the evolution of the sequence.Digestion of mouse satellite DNA with Hae III produces fragments with the same sizes as those produced by Eco RII, but the yields are much lower. It is suggested that Hae III sites have been introduced by divergence and subsequently spread by unequal crossing-over.  相似文献   

16.
F1-ATPase was isolated from yeast S.cerevisiae. The constituent subunits 1 and 2 were purified by gel permeation chromatography, and their amino acid compositions determined. Both subunits have a similar composition except for 12 cystine, methionine, leucine, histidine, and tryptophan. When F1 is treated for three hours with 5′-p-[3H]fluorosulfonylbenzoyl adenosine in dimethylsulfoxide, 90% of the activity is lost. Disc gel electrophoresis of the modified complex showed that over 90% of the label was associated with subunit 2. A labelled peptide from a S.aureus digest of subunit 2 was isolated and sequenced. It had the following amino acid sequence: His-Try1-Asp-Val-Ala-Ser-Lys-Val-Gln-Glu, whereby Tyr1 is the modified amino acid residue. This sequence shows homology to other sequences obtained from maize, beef heart, and E.coli F1-ATPases.  相似文献   

17.
18.
19.
The 1.688 g/cm3 satellite DNA of Drosophila melanogaster is composed primarily of 359 base-pair units repeated in tandem. Most of these units contain a single cleavage site for both HaeIII and HinfI restriction endonucleases; however, some units lack one or both sites. Previously we had shown that the distribution of HaeIII and HinfI endonuclease sites varies widely between different regions of 1.688 g/cm3 satellite DNA; for example, some regions contain HaeIII sites in every unit and other regions (>10,000 base-pairs) contain no HaeIII sites (Carlson &; Brutlag, 1977). We have now cloned molecules of 1.688 g/cm3 satellite DNA which lack HaeIII sites and have shown that the absence of sites is caused by sequence variation rather than base modification. This result indicates that regions of 1.688 g/cm3 satellite DNA with different distributions of restriction sites differ in the sequence of their repeating units. We also show that a large fraction of the satellite DNA which is not cleaved by HaeIII endonuclease still contains HinfI endonuclease sites (and AluI sites) spaced about 359 base-pairs apart. However, one cloned segment lacking HaeIII sites was found to contain 33 tandem copies of a novel 254 base-pair unit. Sequence analysis showed that this 254 base-pair unit is homologous to the 359 repeat except for a 98 base-pair deletion. These data suggest that both units have evolved from a common ancestor and that each has subsequently become amplified into separate tandem arrays.  相似文献   

20.
O6-Methyl[8-3H]deoxyguanosine in a synthetic DNA polymer, poly(dC, dG, m6dG), is demethylated by cell-free extracts of EscherichiacoliBr adapted by exposure to N-methyl-N′-nitro-N-nitrosoguanidine, as shown by the appearance of 3H-labeled deoxyguanosine in hydrolysates of the recovered DNA. The demethylating activity could not be detected in extracts of nonadapted E. coli. These results provide direct evidence that a previously described inducible repair activity in E. coli acts by demethylating O6-methylguanine at the DNA level.  相似文献   

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