Construction and mapping of recombinant plasmids used for the preparation of DNA fragments containing the Escherichia coli lactose operator and promoter.

Three DNA restriction fragments of established sequence containing the Escherichia coli lac genetic controlling regions were cloned. In each case a recombinant plasmid was constructed which was suitable for the subsequent large scale purification of the lac fragment. A 789-base pair HindII fragment, containing the lac operator, promoter, and cyclic AMP receptor protein binding site, was ligated into the single HindII site of the amplifiable plasmid minicolicin E1 DNA (pVH51). A 203-base pair Hae III fragment containing the same genetic sites was ligated into the single Eco RI site of pVH51 which had been "filled in" by the Micrococcus luteus DNA polymerase. Thus, the lac fragment was inserted between two Eco RI sites. Plasmids containing multiple copies of this Eco RI fragment were then constructed. A 95-base pair Alu I fragment containing the lac promoter and operator was cloned similarly. Also, the 203-base pair fragment was cloned into the Eco RI site of pVH51 using a 300-base pair linker fragment (isolated by RPC-5 column chromatography) which permitted retention of its Hae III ends. Mapping studies on pVH51 DNA with a number of DNA restriction endonucleases, including Alu I, Taq I, and Hpa II, are described.     

DNA fragments associated with chromosome scaffolds

Following extensive digestion of HeLa metaphase chromosomes with either Hae III endonuclease or micrococcal nuclease, nonhistone protein scaffolds may be isolated. Scaffolds isolated after Hae III digestion have about 1.5% of the chromosomal DNA attached to them. This DNA is heterogeneous in size, ranging from about 0.2 to 20 kbp. It can be cleaved with either Eco RI or Hae III - Eco RI, producing a series of repeated fragments, of which the most abundant is 1.7 kbp in length. The 1.7-kdp fragment is tandemly repeated and is enriched (about 50-fold) in the scaffold-associated DNA. It is located primarily on human chromosome 1 and is probably a component of human satellites II and III. Scaffolds isolated after micrococcal nuclease digestion have about 0.1% of chromosomal DNA attached. This DNA is present in two size classes - fragments larger than 10 kbp and fragments approximately 0.2 kbp long. Restriction enzyme digestion of this DNA gives no prominent repeated fragments. Its reassociation kinetics are similar to those of total DNA, indicating that it is not enriched in either highly repetitive or middle repetitive sequences.     

A METHOD FOR the DETERMINATION of STRANDEDNESS of DNA FRAGMENTS CLONED IN PHAGE M13

Recombinant M13Hol phage containing Eco R1 restriction endonuclease fragments B, E, and F of adenovirus type 2 (Ad2) DNA were constructed by cloning into the unique Eco R1 site of the replicative form of the phage M13Hol176 DNA. Polarity of the adenovirus inserts in recombinant molecules was deduced by the following procedures: Viral DNA fragments obtained from Ad2 DNA molecules were purified, denatured, and subjected to electrophoresis. the separated DNA strands were transferred from agarose to nitrocellulose by the Southern procedure and hybridized with radioactive 3'-end labeled Hae III fragments of the recombinant phage DNAs. This procedure provided a rapid test for assaying strandedness of the cloned fragments.     

Organization and chromosomal specificity of autosomal homologs of human Y chromosome repeated DNA

The human Y chromosome contains a group of repeated DNA elements, identified as 3.4-kilobase pair (kb) fragments in Hae III digests of male genomic DNA, which contain both Y-specific and non-Y-specific sequences. We have used these 3.4-kb Hae III Y fragments to explore the organizational properties and chromosomal distribution of the autosomal homologs of the non-Y-specific (NYS) 3.4-kb Hae III Y elements. Three distinct organizations, termed domains, have been identified and shown to have major concentrations on separate chromosomes. We have established that domain K is located on chromosome 15 and domain D on chromosome 16 and suggested that domain R is on chromosome 1. Our findings suggest that each domain is composed of a tandemly arrayed cluster of a regularly repeating unit containing two sets of repeated sequences: one that is homologous to the NYS 3.4-kb Hae III Y sequences and one that does not cross-react with the 3.4-kb Hae III Y repeats. Thus, these autosomal repeated DNA domains, like their Y chromosome counterparts, consist of a complex mixture of repeated DNA elements interspersed among each other in ways that lead to defined periodicities. Although each of the three identified autosomal domains cross-reacts with 3.4-kb Hae III Y fragments purified from genomic DNA, the length periodicities and sequence content of the autosomal domains are chromosome specific. The organizational properties and chromosomal distribution of these NYS 3.4-kb Hae III homologs seem inconsistent with stochastic mechanisms of sequence diffusion between chromosomes.     

Long range periodicities in mouse satellite DNA.

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.     

Segments of mitochondrial DNA of yeast carrying antibiotic resistances

Summary Mitochondrial DNA was isolated from an oligomycin-resistant petite mutant of yeast, Saccharomyces cerevisiae. It had repeated sequences of 3600 base pairs. This segment was about one twentieth of the whole mtDNA of wild type yeast, which had a size of 74 kilo base pairs.This segment of mtDNA had one cleavage site for a restriction endonuclease, Hind II, which was more resistant to cleavage than the other Hind II sites in wild type mtDNA. It had two cleavage sites for Hha I and gave two Hha fragments, which were arranged alternatively. Digestion with Hae III gave four fragments and these fragments were mapped.Mitochondrial DNA of this mutant showed a loss of heterogeneity in a melting profile. It melted within a narrow range of temperature, which was similar to that of poly dA·poly dT. Its differential melting curve was significantly different from that of wild type mtDNA.Mapping of mtDNA of a wild type yeast was carried out with restriction endonucleases. Fragments of mtDNA, which were isolated from petites carrying oligomycin-erythromycin-chloramphenicol-resistance and erythromycin-chloramphenicol resistance were also mapped. Loci of oligomycin-resistance, erythromycin-resistance and chloramphenicol-resistance were investigated based on the maps of Eco R I fragments and Hind II fragments.     

Multiple structures of adeno-associated virus DNA: analysis of terminally labeled molecules with endonuclease R-Hae III.

The double-stranded form of adeno-associated virus (AAV) DNA has about 20 sites sensitive to endonuclease R.Hae III from Haemophilus aegypitus; the fragments produced fall into about 13 size classes, 8 of which contain single fragments. The location of the Hae III-produced AAV fragments relative to the three EcoR1 fragments was determined. Using revised figures for the molecular weights of the Hae III cleavage products of phiX174 replicative form DNA, we calculated that AAV DNA contains about 4,000 nucleotides. After Hae III digestiion of duplex DNA terminally labeled with 32P using polynucleotide kinase, the majority of fragments containing a 5' 32P label were about 40 nucleotides in length, and fragments of similar size were generated from each end, suggesting that the Hae site closest to the end is within the terminal repetition. Two more-slowly-migrating cleavage products also bore 5' 32P end label. These three terminally labeled species were also generated from single-stranded AAV DNA by digestion with Hae III, and evidence that one may have a nonlinear ("rabbit-ear") structure is presented. The predominant 5' terminal base was identified as thymine for both the plus and minus strands of AAV. Single-stranded AAV molecules could not be efficiently covalently circularized by incubation with polynucleotide ligase or ligase plus T4 DNA polymerase.     

A stretch of "late" SV40 viral DNA about 400 bp long which includes the origin of replication is specifically exposed in SV40 minichromosomes.

Examination of DNA fragments produced from either formaldehyde-fixed or unfixed SV40 minichromosomes by multiple-cut restriction endonucleases has led to the following major results: Exhaustive digestion of unfixed minichromosomes with Hae III generated all ten major limit-digest DNA fragments as well as partial cleavage products. In striking contrast to this result, Hae III acted on formaldehyde-fixed minichromosomes to yield only one of the limit-digest fragments, F, which is located in the immediate vicinity of the origin of replication, spanning nucleotides 5169 and 250 on the DNA sequence map of Reddy et al. (1978). This 300 base pair (bp) fragment was released as naked DNA from formaldehyde-fixed, Hae III-digested minichromosomes following treatment either by pronase-SDS or by SDS alone. In the latter case, the remainder of the minichromosome retained its compact configuration as assayed by both sedimentational and electrophoretic methods. In minichromosomes, the F fragment is therefore not only accessible to Hae III at its ends, but is also neither formaldehyde cross-linked into any SDS-resistant nucleoprotein structure nor topologically "locked" within the compact minichromosomal particle. This same fragment was preferentially produced during the early stages of digestion of unfixed minichromosomes with Hae III, and its final yield in the exhaustive Hae III digest was significantly higher than that of other limit-digest fragments. Similar results were obtained upon digestion of either unfixed or formaldehyde-fixed minichromosomes with Alu I. In particular, of approximately twenty major limit-digest DNA fragments, only two fragments (F and P, encompassing nucleotides 5146 to 190, and 190 to 325, respectively) were produced by Alu I from the formaldehyde-fixed minichromosomes. All other restriction endonucleases tested (Mbo I, Mbo II, Hind III, Hin II+III and Hinf I), for which there are no closely spaced recognition sequences in the above mentioned regions of the SV40 genome, did not produce any significant amount of limit-digest DNA fragments from formaldehyde-fixed minichromosomes. These findings, taken together with our earlier data on the preferential exposure of the origin of replication in SV40 minichromosomes (Varshavsky, Sundin and Bohn, 1978), strongly suggest that a specific region of the "late" SV40 DNA approximately 400 bp long is uniquely exposed in the compact minichromosome. It is of interest that, in addition to the origin of replication, this region contains binding sites for T antigen (Tjian, 1977), specific tandem repeated sequences and apparently also the promoters for synthesis of late SV40 mRNAs (Fiers et al., 1978; Reddy et al., 1978).     

The ovalbumin gene: alleles created by mutations in the intervening sequences of the natural gene.

Two allelic forms of the natural chicken ovalbumin gene have been independently cloned. These alleles differ from each other by an Eco RI restriction cleavage site in one of the seven intervening sequences within the natural ovalbumin gene. Restriction endonuclease mapping and sequence analyses of these cloned genotypic alleles have shown identical sequence organization and molecular structures of the interspersed structural and intervening sequences except for the particular Eco RI cleavage site. Sequencing data of the cloned DNA suggest that this Eco RI site may be created or eliminated by a single base mutation in the intervening sequence of the ovalbumin gene. The occurrence of apparent homozygous and heterozygous allelic forms of the ovalbumin gene in individual hens and roosters within the same breed has been observed. 10 and 40% of the chickens examined are homozygous for the ovalbumin gene with and without the extra Eco RI site, respectively, while 50% of them are heterozygous. Further analysis of individual chicken DNA cleaved by restriction endonuclease Hae III has revealed that there may be a series of such mutational variations within the ovalbumin gene. We have identified two Hae III cleavage sites that do not occur in all of the chickens, thus giving rise to several additional allelic variations of the ovalbumin gene. At least one of these Hae III sites is situated in the intervening sequence of the ovalbumin gene, and its lcoation has been mapped. Such allelic variations must be taken into consideration when determining eucaryotic gene structure by restriction mapping of the genomic DNA. Furthermore, this type of mutation within the intervening sequences of an eucaryotic gene has no known phenotypic manifestation. It represents an extrastructural silent mutation that must be taken account of in studies to estimate the rates of eucaryotic gene sequence divergence during evolution.     

Physical map of Aspergillus nidulans mitochondrial genes coding for ribosomal RNA: an intervening sequence in the large rRNA cistron

Summary A detailed map of the 32 kb mitochondrial genome of Aspergillus nidulans has been obtained by locating the cleavage sites for restriction endonucleases Pst I, Bam H I, Hha I, Pvu II, Hpa II and Hae III relative to the previously determined sites for Eco R I, Hind II and Hind III. The genes for the small and large ribosomal subunit RNAs were mapped by gel transfer hybridization of in vitro labelled rRNA to restriction fragments of mitochondrial DNA and its cloned Eco R I fragment E3, and by electron microscopy of RNA/DNA hybrids.The gene for the large rRNA (2.9 kb) is interrupted by a 1.8 kb insert, and the main segment of this gene (2.4 kb) is separated from the small rRNA gene (1.4 kb) by a spacer sequence of 2.8 kb length.This rRNA gene organization is very similar to that of the two-times larger mitochondrial genome of Neurospora crassa, except that in A. nidulans the spacer and intervening sequences are considerably shorter.     

Heterogeneity in Chinese hamster ribosomal DNA

A discrete heterogeneity has been detected in Chinese hamster ribosomal DNA after Eco R1 digestion of total DNA followed by a Southern transfer and hybridization with [¹²⁵I]18S or [¹²⁵I]28S ribosomal RNA. Digestion with Eco R1 produces three fragments, 4.3, 6.0 and 9.5×10⁶ daltons respectively, which hybridize with 18S RNA. The smallest fragment also hybridizes with 28S RNA. Either length heterogeneity or sequence heterogeneity (i.e. presence of an additional Eco R1 site in some of the rDNA molecules) must be invoked to account for the two larger Eco R1 fragments that contain 18S but not 28S sequences. Eco R1 and Hind III maps, consistent with either length or sequence heterogeneity, are presented. The data at this time, however, do not distinguish between the two alternatives.     

Cleavage patterns of Drosophila melanogaster satellite DNA by restriction enzymes.

The five satellite DNAs of Drosophila melanogaster have been isolated by the combined use of different equilibrium density gradients and hydrolyzed by seven different restriction enzymes; Hae III, Hind II + Hind III, Hinf, Hpa II, EcoR I and EcoR II. The 1.705 satellite is not hydrolyzed by any of the enzymes tested. Hae III is the only restriction enzyme that cuts the 1.672 and 1.686 satellites. The cleavage products from either of these reactions has a heterogeneous size distribution. Part of the 1.688 satellite is cut by Hae III and by Hinf into three discrete fragments with M.W. that are multiples of 2.3 X 10(5) daltons (approximately 350 base pairs). In addition, two minor bands are detected in the 1.688-Hinf products. The mole ratios of the trimer, dimer and monomer are: 1:6.30 : 63.6 for 1.688-Hae III and 1 : 22.0 : 403 for 1.688-Hinf. Circular mitochondrial DNA (rho = 1.680) is cut into discrete fragments by all of the enzymes tested and molecular weights of these fragments have been determined.     

Mapping of mitochondrial DNA of individual sheep and goats: Rapid evolution in the D loop region

Mitochondrial DNA (mtDNA) from sheep and goat was compared by restriction endonuclease analysis and heteroduplex mapping in the electron microscope. The fragment patterns produced by endonuclease Hae III from three individual sheep and two goat mtDNAs all differed from each other. The three sheep mtDNAs had identical Eco RI and Hind III fragments, but the two goat mtDNA patterns differed from each other as well as from sheep mtDNA. We estimate that each sheep mtDNA differs from each other by 0.5–1% of its nucleotide sequences, the two goat mtDNAs by 1–2%, and there is a 6–11% sequence difference between sheep and goat mtDNAs. We have mapped the Eco RI and Hind III sites of goat and sheep mtDNA and determined the positions of the D loop, which marks the replication origin, relative to the restriction map. The D loops are at homologous positions on the mtDNAs from both species, but the goat D loop is only 75% as long as the sheep D loop. Regions with a high degree of sequence divergence occur at both ends of the D loop. We suggest that a duplication of about 150 base pairs has occurred in the region where the sheep and goat D loops differ in length. We discuss mtDNA evolution in terms of divergence of isolated “mitochondrial DNA clones.”     

The banding pattern produced by restriction endonucleases in mouse chromosomes

The metaphase chromosomes of mouse have been treated with the restriction endonucleases Alu I, Mbo I, Hae III and Eco RII and subsequently stained with the DNA-specific dye Ethidium bromide. A striking correspondence between previous biochemical findings and our cytological results has been noticed with Alu I, Mbo I and Hae III, which were capable of digesting all but the DNA localized at the centric constitutive heterochromatic areas. Digestion with Eco RII, on the contrary, resulted in cytological data which apparently did not fit in with the biochemical results previously obtained by digesting the DNA of this species with the same enzyme. It is postulated that factors such as chromatin organization, in addition to DNA base sequence, can determine the results we report.     

Diversity of rat strains and tumor lines of DNA fragments homologous to an amplified 5.8 kilobase ECO R1 fragment of Novikoff hepatoma cells

The nuclear DNA of several different rat strains and rat tumor lines have been analyzed with respect to Eco R1 fragments homologous to the amplified 5.8 kb Eco R1 fragment (fragment A) of Novikoff hepatoma cells. Two Eco R1 fragments, 4.8 and 4.4 kb, which hybridized to the 5.8 kb Eco R1 fragment, were found in all the genomes investigated. Although none of the examined genomes exhibited evidence of the same degree of amplification of fragment A related sequences as that of Novikoff hepatoma, several had Eco R1 fragments of various other sizes which were homologous to fragment A. These results indicate that the family of fragment A homologous sequences consists of two populations, the constant 4.8 and 4.4 kb fragments, and a second group of sequences which varies with respect to size.     

Mitochondrial DNA from Podospora anserina. II. Properties of mutant DNA and multimeric circular DNA from senescent cultures.

Summary Mitochondrial (Mt) DNA from mitochondrial mutants of race s Podospora anserina and from senescent cultures of races s and A was examined. In mutants, we observed that fewer full length circles (31 ) were present; instead, smaller circles characteristic for each mutant sudied were found. Eco Rl digestion of these mutant MtDNAs indicated that in certain mutants, although specific fragments were absent, the total molecular weight of the fragments was not much different than wild-type.The properties of senescent MtDNA was strikingly different from either wild-type or mutant Mt DNA. First, a multimeric set of circular DNA was observed for both race s and A, with a monomeric repeat size of 0.89 . These circles ranged in size from 0.89 to greater than 20 ; only one molecule out of some 200 molecules was thought to be of full length (31 ). Density gradient analysis showed that there were two density species: a majority were at the same density as wild-type (1.694 g/cm³) and a second at 1.699 g/cm³. Most of the circular molecules from MtDNA isolated by either total DNA extraction or by extraction of DNA from isolated mitochondria were contained in the heavy DNA fraction. Eco R1 enzymatic digestion indicated that the light DNA had several fragments (amounting to about 23×10⁶ daltons) missing, compared with young, wild-type MtDNA. Heavy senescent MtDNA was not cleaved by Eco R1. Analysis with Hae III restriction endonuclease showed also that light senescent MtDNA was missing certain fragments. Heavy MtDNA of average size 20×10⁶ daltons, yielded only one fragment, 2,500 bp long, by digestion with Hae III restriction endonuclease. Digestion of heavy DNA with Alu I enzyme yielded 10 fragments totalling 2,570 bp. By three criteria, electron-microscopy, Eco R1 and Hae digestion, we conclude that the heavy MtDNA isolated from senescent cultures of Podospora anserina consisted of a monomeric tandemly repeating subunit of about 2,600 bp length.These results on the properties of senescent MtDNA are discussed with regard to the published properties of the rho ^- mutation in the yeast, S. cerevisiae.     

A single cleavage of Simian virus 40 (SV40) DNA by a site specific endonuclease from Thermus aquaticus, Taq I.

A site specific endonuclease from Thermus aquaticus, Taq I, cleaves Simian virus 40 (SV40) DNA at a single site. The cleavage site was localized on the physical map by double digestions, using the previously characterized fragments produced by digestion with Hae II, Hae III, AluI, HhaI, HinfI, or BstI. The Taq I site is located at the position that is 56.5% of the unit length from the Eco RI site.     

The leftward promoter of bacteriophage lambda. Isolation on a small restriction fragment and deletion of adjacent regions

As part of our investigations on the relationship between DNA structure and gene regulation, a 352-base pair Hae III fragment was cloned containing the leftward operator-promoter (PL) region of bacteriophage lambda. This was accomplished without the aid of a phenotypic assay for the cloned fragment. A Hae III digest of a segment of the lambda genome was first fractionated by RPC-5 column chromatography. The partially purified PL fragment was then ligated into the Eco RI site of the pBR322 plasmid vector and cloned into the recBC+ Escherichia coli host C600(R-M-) using a technique that converts the Hae III ends of the fragment into Eco RI sites. Similar cloning attempts into a recBC- host (C600-SF8) were unsuccessful. The cloned fragment has the PL promoter oriented toward the tetracycline resistance genes of the vector, and is isolated from the plasmid (pRW601) by digestion with Eco RI followed by sucrose gradient sedimentation. The fragment was identified as PL by restriction mapping, repressor binding, sequencing, and promoter location. The now complete sequence of this fragment, part of which was known previously, reveals a large A/T-rich region immediately adjacent to the PL promoter. We have generated deletions in this region in order to study the influence of this sequence on promoter function.     

DNA fragments of 300 base pairs released from metaphase chromosomes by digestion with deoxyribonuclease I

DNase I digestion of metaphase chromosomes, that have been extensively digested with Hae III, further released chromosomal DNA and proteins; 3.3% and 10.8% of the chromosomal DNA and proteins, respectively, remained insoluble. However, digestion of chromosomes first with DNase I followed by Hae III caused most of the proteins to remain in the insoluble fraction. DNase I released DNA fragments of 300 base pairs long which were not released by Hae III digestion. These DNA fragments may be protected by protein components from further fragmentation by DNase I.