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1.
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   

2.
Isolation of a multigene family containing human alpha-tubulin sequences   总被引:6,自引:0,他引:6  
The boundaries of the origin of polyoma DNA replication have been analyzed using a set of deletion mutants. The majority of these had small deletions, 5 to 30 base-pairs in size, which together removed most of the non-translated sequences of the genome. The phenotype of the mutants was characterized by analysis of infectivity, transforming ability and DNA synthesis. All mutants with reduced or abolished infectivity had corresponding defects of viral DNA synthesis. The effect of the deletion was cis-acting, since the replication of the mutants was not stimulated by the presence of wild-type DNA. Deletions causing a reduction of DNA synthesis were found at two sites. The first at the 32 base-pair inverted repeat sequence and the neighbouring A · T tract previously implicated in the initiation of DNA synthesis, and the second close to the late genes. The two sites were separated by at least 60 base-pairs of non-essential DNA. Only one mutant with a deletion at the second site was unable to express early gene functions.The mutants were constructed by linearization, shortening and recircularization of polyoma DNA inserted into the plasmid pBR322. The mutagenesis was directed at restriction endonuclease BglI or PvuII cleavage sites. The BglI-directed mutagenesis was focussed to polyoma DNA by using as a vector a derivative of pBR322 resistant to cleavage by BglI.  相似文献   

3.
The nucleotide sequence running from the genetic left end of bacteriophage T7 DNA to within the coding sequence of gene 4 is given, except for the internal coding sequence for the gene 1 protein, which has been determined elsewhere. The sequence presented contains nucleotides 1 to 3342 and 5654 to 12,100 of the approximately 40,000 base-pairs of T7 DNA. This sequence includes: the three strong early promoters and the termination site for Escherichia coli RNA polymerase: eight promoter sites for T7 RNA polymerase; six RNAase III cleavage sites; the primary origin of replication of T7 DNA; the complete coding sequences for 13 previously known T7 proteins, including the anti-restriction protein, protein kinase, DNA ligase, the gene 2 inhibitor of E. coli RNA polymerase, single-strand DNA binding protein, the gene 3 endonuclease, and lysozyme (which is actually an N-acetylmuramyl-l-alanine amidase); the complete coding sequences for eight potential new T7-coded proteins; and two apparently independent initiation sites that produce overlapping polypeptide chains of gene 4 primase. More than 86% of the first 12,100 base-pairs of T7 DNA appear to be devoted to specifying amino acid sequences for T7 proteins, and the arrangement of coding sequences and other genetic elements is very efficient. There is little overlap between coding sequences for different proteins, but junctions between adjacent coding sequences are typically close, the termination codon for one protein often overlapping the initiation codon for the next. For almost half of the potential T7 proteins, the sequence in the messenger RNA that can interact with 16 S ribosomal RNA in initiation of protein synthesis is part of the coding sequence for the preceding protein. The longest non-coding region, about 900 base-pairs, is at the left end of the DNA. The right half of this region contains the strong early promoters for E. coli RNA polymerase and the first RNAase III cleavage site. The left end contains the terminal repetition (nucleotides 1 to 160), followed by a striking array of repeated sequences (nucleotides 175 to 340) that might have some role in packaging the DNA into phage particles, and an A · T-rich region (nucleotides 356 to 492) that contains a promoter for T7 RNA polymerase, and which might function as a replication origin.  相似文献   

4.
5.
A fine-structure physical and genetic map was constructed of a 1000 base-pair region of the l-arabinose operon of Escherichia coli. This region consists of the ara regulatory sequences contained between the araC and araB genes and portions of these flanking genes. Point mutations, Mu phage insertions, and bacterial deletions as well as arabinose-induced and basal enzyme levels in the strains were used in constructing a genetic map of the region. These ordered positions were then located more accurately by mapping the point mutations against physically located endpoints of deletions isolated on the two non-defective transducing phage λparaB114 and λparaC116. Phage possessing deletions ending in the arabinose regulatory region were isolated from indicating-plates on which deletions removing none, part, or all of either the araC or araB genes carried on the phage could be distinguished. Phage stocks were enriched for such deletions prior to plating by treatment with chelating agents and heat (Parkinson &; Huskey, 1971). Deletions into the ara region on either phage shorten the ara DNA homology region formed from heteroduplexes between λparaB114 and λparaB116. Therefore the physical locations of these deletion endpoints were determined by electron microscopy of the appropriate heteroduplexes and/or by gel electrophoresis of the central duplex following S1 nuclease digestion (Lis &; Schleif, 1975b). 18 of the 32 deletions isolated and mapped in this region were measured physically. The space between araC and araB, containing the regulatory elements of the operon, is estimated to be about 300 base-pairs.  相似文献   

6.
The precise positions of the origin of replication3 and of the D-loop within the HpaII restriction map of HeLa cell mitochondrial DNA have been investigated. For this purpose, 7 S DNA, which is the heavy-chain initiation sequence, was used as a template for fragment-primed DNA synthesis by Escherichia coli DNA polymerase I. The results indicate clearly that the origin of replication lies in HpaII fragment 8 at about 80 base-pairs from the border with fragment 17, and that the D-loop region extends from this site, through fragment 17, to a position in fragment 10 which is about 365 base-pairs from the border with fragment 17. Sequential digestion of fragment 8 with HaeIII enzyme has allowed the isolation of a subfragment, about 200 base-pairs long, that contains the origin of replication.  相似文献   

7.
An 873 base-pair DNA sequence from the rII region of bacteriophage T4 is presented. The sequence encodes 139 carboxyl-terminal amino acids of rIIA and the amino-terminal 146 amino acids of rIIB. Eleven base-pairs separate the rIIA stop codon (UAA) and the rIIB AUG.An extensive genetic map is superimposed on the DNA sequence, showing the deduced locations of many of the mutations (base-pair substitutions, frameshifts, deletions) found in previous rII genetic studies.  相似文献   

8.
A 1.7 × 103 base-pair SalI fragment of mouse ribosomal gene spacer undergoes recA-independent deletions of DNA in units of approximately 126 base-pairs when cloned in λ or bacterial plasmids. When we examined the structure of the 1.7 × 103 base-pair piece with PvuII we found it to be composed of about equal numbers of copies of each of two subrepeating units, 120 and 130 base-pairs in size. The correlation between the size of the structural subunits and the functional genetic unit of this fragment as expressed in Escherichia coli led us to study the organization of these sequences in mice. SalI (or HindII) digests of DNA samples from wild and inbred strains revealed extensive heterogeneity in the size of fragments homologous to this 1.7 × 103 base-pair piece. A total of 15 different size classes were detected in our samples. We found that these fragments were also organized in PvuII repeating units about equal in size to the PvuII repeats in the cloned 1.7 × 103 base-pair piece. Using an objective analytical procedure (see the Appendix) we determined that the 15 different fragments found in our mouse DNA samples probably originated as a result of genetic events based on a 135 base-pair structural unit.We consider the similarity between the size of the PvuII structural unit and the unit of genetic behavior in both the cloned and uncloned DNA samples to be significant. We suspect that there are aspects of the nucleotide structure or organization of the PvuII repeating units that play a dominant role in its genetic behavior, regardless of whether these sequences are present in E. coli or mice. We believe that the clones containing this mouse sequence may provide an experimental system for studying the nature of the genetic events that are involved in multigene evolution.  相似文献   

9.
X-ray-induced mitotic recombination rates and spontaneous meiotic recombination rates have been determined in two-point crosses of various defined cyc1 mutants of the yeast Saccharomyces cerevisiae. All but one of the 17 cyc1 mutants chosen for this study contained either the addition, deletion or substitution of single base-pairs located within a defined segment of the gene that corresponds to the 11 amino acid residues at the amino terminus of iso-1-cytochrome c; approximately half of these mutants had alterations of the AUG initiation codon, some at the same base pair. Up to 66-fold differences in X-ray-induced recombination rates were observed when the same cyc1 mutant was crossed to cyc1 mutants having different alterations in the AUG initiation codon; over a ten-fold difference was observed in series of homologous crosses involving mutants with different changes at the same base-pair. Recombination rates that were associated with specific cyc1 mutants co-segregated with the particular alleles following meiosis, and comparable recombination patterns were also observed for independently isolated, identical mutations. With the mutants used in this study, the frequencies of meiotic recombination did not differ as markedly, suggesting a dissimilar dependence on specific DNA sequences for these two modes of recombination. These disproportionalities of recombination rates suggest that the nature of the mismatched bases influences the recombination process, but not in a way that can be simply interpreted.  相似文献   

10.
Summary DNA molecules of seven T7 mutants with overlapping deletions in the early region were cleaved by restriction enzymes HindII, HpaI and II, and HaeIII. The differences in the cleavage patterns after electrophoresis have been used to generate a cleavage map of the restriction sites of this enzyme. It covers the first 9% of the T7 DNA molecule. Cleavage points for HindII are at 0.60, 1.33, 1.59, 1.76, 5.26, 6.27, 7.4 and 8.38%; for HpaI and II at 1.36, 1.62, 4.46, 6.29, 6.62, 7.56, and 8.76%; for HaeIII at 3.85, 6.98, 7.88 and 8.26%. Some fragments have been located in the region containing the early promoters, others carry the complete sequences of gene 0.3.  相似文献   

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