DNA sequence variants in the G gamma-, A gamma-, delta- and beta-globin genes of man

DNA prepared from 60 unrelated individuals was cleaved with one of eight different restriction endonucleases and the resulting DNA fragments were separated by agarose gel electrophoresis. DNA fragments containing G gamma-, A gamma-, delta- or beta-globin genes were detected by Southern blot hybridization, using as probe either a 32P-labeled cloned DNA copy of rabbit beta-globin messenger RNA or labeled human beta- and G gamma- globin cDNA plasmids. Three types of variant restriction enzyme patterns of globin DNA fragments were detected in otherwise normal individuals. One variant pattern, found in only one person, was caused by an additional restriction endonuclease Pst I cleavage site in the center of the delta- globin gene intervening sequence; the subject was heterozygous for the presence of this cleavage site and was shown to have inherited it from her mother. Another variant pattern resulted from the appearance of an endonuclease Hind III cleavage site in the intervening sequence of the A gamma-globin gene; this variant is polymorphic, with a gene frequency for the presence of the intragenic Hind III site of 0.23. This Hind III cleavage site polymorphism is also found in the G gamma-globin gene intervening sequence and thus the polymorphism itself appears to be duplicated over the pair of gamma-globin loci. These variants can be used to derive an approximate estimate of the total number of different DNA sequence variants in man.     

Physical map of the bacteriophage T5 genome based on the cleavage products of the restriction endonucleases SalI, SmaI, BamI, and HpaI.

A physical map of the bacteriophage T5 genome was constructed by ordering the fragments produced by cleavage of T5 DNA with the restriction endonucleases SalI (4 fragments), SmaI (4 fragments), BamI (5 fragments), and HpaI (28 fragments). The following techniques were used to order the fragments. (i) Digestion of DNA from T5 heat-stable deletion mutants was used to identify fragments located in the deletable region. (ii) Fragments near the ends of the T5 DNA molecule were located by treating T5 DNA with lambda exonuclease before restriction endonuclease cleavage. (iii) Fragments spanning other restriction endonuclease cleavage sites were identified by combined digestion of T5 DNA with two restriction endonucleases. (iv) The general location of some fragments was determined by isolating individual restriction fragments from agarose gels and redigesting the isolated fragments with a second restriction enzyme. (v) Treatment of restriction digests with lambda exonuclease before digestion with a second restriction enzyme was used to identify fragments near, but not spanning, restriction cleavage sites. (vi) Exonucleases III treatment of T5 DNA before restriction endonuclease cleavage was used to locate fragments spanning or near the natural T5 single-chain interruptions. (vii) Analysis of the products of incomplete restriction endonuclease cleavage was used to identify adjacent fragments.     

Rapid mapping of transposon insertion and deletion mutations in the large Ti-plasmids of Agrobacterium tumefaciens.

A procedure is presented, that has allowed the rapid assignment of transposon Tn1 and Tn7 insertion sites in the large (130 Md) nopaline Ti-plasmid pTiC58, to specific restriction enzyme fragments. Total bacterial DNA is isolated from Agrobacterium tumefaciens strain C58 mutants that carry a transposon in their Ti-plasmid, and digested with an appropriate restriction endonuclease. The fragments are separated on an agarose gel, denatured and transferred to nitrocellulose filters. These are hybridized against purified wild type pTiC58, or against segments of PTiC58, cloned in E. coli using pBR322 as a vector plasmid. DNA sequences homologous to the probe are detected by autoradiography, thus generating a restriction enzyme pattern of the plasmid from a digest of total bacterial DNA. Mutant fragments can be readily identified by their different position compared to a wild type reference. This protocol eliminates the need to separate the large plasmid from chromosomal DNA for every mutant. In principle, it can be applied to the restriction enzyme analysis of insertion or deletion mutants in any plasmid that has no extensive homology with the chromosome.     

Isolation of deletion and substitution mutants of adenovirus type 5

The infectivity of adenovirus type 5 DNA can be increased to about 5 x 10³ plaque-forming units per μg DNA if the DNA is isolated as a DNA-protein complex. Utilizing this improved infectivity, a method was developed for the selection of mutants lacking restriction endonuclease cleavage sites. The procedure involves three steps. First, the DNA-protein complex is cleaved with a restriction endonuclease. The Eco RI restriction endonuclease was used here. It cleaves adenovirus type 5 DNA to produce three fragments: fragment A (1–76 map units), fragment C (76–83 map units) and fragment B (10–83 map units). Second, the mixture of fragments is rejoined by incubating with DNA ligase, and, third, the modified DNA is used to infect cells in a DNA plaque assay. Mutants were obtained which lacked the endonuclease cleavage site at 0.83 map units. Such mutant DNAs were selected by this procedure because they were cleaved by the Eco RI endonuclease to produce only two fragments: a normal A fragment and a fused B/C fragment. These two fragments could be rejoined to produce a viable DNA molecule as a result of a bimolecular reaction with one ligation event; this exerted a strong selection for such molecules since a trimolecular reaction (keeping the C fragment in its proper orientation) and two ligation events were required to regenerate a wild-type molecule. The alterations resulting in the loss of the Eco RI endonuclease cleavage site at 0.83 map units include both deletion and substitution mutations. The inserted sequences in the substitution mutations are cellular in origin.     

In vivo cleavage of cytosine-containing bacteriophage T4 DNA to genetically distinct, discretely sized fragments

Mutants of bacteriophage T4D that are defective in genes 42 (dCMP hydroxymethylase), 46 (DNA exonuclease), and 56 (dCTPase) produce limited amounts of phage DNA in Escherichia coli B. In this DNA, glucoylated 5-hydroxymethylcytosine is completely replaced by cytosine. We found that this DNA rapidly becomes fragmented in vivo to at least 16 discrete bands as visualized on agarose gels subjected to electrophoresis. The sizes of the fragments ranged from more than 20 to less than 2 kilobase pairs. When DNAs from two of these bands were radioactively labeled in vitro by nick translation and hybridized to XbaI restriction fragments of cytosine-containing T4 DNA, evidence was obtained that the two bands are genetically distinct, i.e., they contain DNA from different parts of the T4 genome. Mutational inactivation of T4 endonuclease II (gene denA) prevented the fragmentation. Three different mutations in T4 endonuclease IV (gene denB) caused the same minor changes in the pattern of fragments. We conclude that T4 endonuclease II is required, and endonuclease IV is involved to a minor extent, in the in vivo production of these cytosine-containing T4 DNA fragments. We view these DNA fragments as "restriction fragments" since they represent degradation products of DNA "foreign" to T4, they are of discrete size, and they are genetically distinct. Thus, this report may represent the first, direct in vivo demonstration of discretely sized genetically distinct DNA restriction fragments.     

Expression of unselected adenovirus genes in human cells co-transformed with the HSV-1 tk gene and adenovirus 2 DNA

We have introduced adenovirus 2 genes into high molecular weight DNA of permissive human cells by co-transformation of tk- human 143 cells with Ad2 restriction enzyme fragments and a cloned Bam HI fragment that carries the HSV-1 thymidine kinase gene. Tk+ cells were isolated after selection and maintenance in HAT medium. Several co-transformed lines are able to complement the growth of Ad5 dl312 (delta 1.2--3.7) and Ad5 dl434 (delta 2.6--8.7), deletion mutants that lack sequences from the left end of the viral genome. The amount and arrangement of viral sequences in the co-transformed cell lines have been analyzed by restriction endonuclease digestion and filter hybridization. Most of the cell lines contain a single insertion of the HSV-1 tk fragment and a single insert of adenoviral DNA. However, one line (B1) contains at least four different insertions, two of which are present in multiple copies. The adenoviral DNA in all cell lines is composed of sequences from the left end of the genome and extends for varying lengths in different lines. Two cell lines that complement deletion mutants efficiently synthesize both early region 1a and 1b mRNAs. The B1 line synthesizes low levels of 1a mRNA, higher levels of 1b mRNA and a unique mRNA that maps to the right of the 1b gene family. When grown continuously in HAT medium, some cell lines are quite stable while others are fairly unstable. Some tk+ subclones support the growth of viral mutants as well as the parental line while others give reduced levels of complementation. For all tk+ subclones examined, the alteration or reduction in viral gene expression is independent of changes in the pattern of integration of viral DNA.     

Construction of a genome library from a beta-0-thalassemic individual from Ferrara: characterization of clones containing beta globin genes

A library of genomic DNA was prepared from a patient with beta o Ferrara thalassaemia: random human DNA fragments (15 - 20 Kb) have been joined to phage lambda vectors and cloned has viable phage particles (4). 4x10(5) phages have been screened for their content in beta globin gene sequences, using a human beta cDNA plasmid (5) as hybridization probe. Five positive clones have been isolated and characterized by restriction endonuclease cleavage analysis and by the hybridization experiments. The results obtained allow the precise localization of the human fragments inside the beta like globin gene cluster (6). The comparison of the thalassaemic fragments with the normal DNA (6 - 7) shows two different restriction endonuclease sites, for Xba I and Eco RI respectively, downstream from the human beta globin gene.     

Organization of the yeast ribosomal RNA gene cluster via cloning and restriction analysis.

The restriction endonuclease EcoR1 cleaves Saccharomyces cerevisiae DNA, which codes for ribosomal RNA (rRNA), into seven fragments, A second restriction endonuclease, HindIII, cleaves the same yeast ribosomal DNA into two fragments. These two restriction enzymes each yield DNA segments that total about 5.9 megadaltons. The "repeat unit" of the yeast genes coding for rRNA is thus about 5.9 megadaltons or about 9000 base pairs long. The two HindIII-cleaved DNA fragments as well as one of the EcoR1-cleaved DNA fragments were purified and amplified by cloning in Escherichia coli. Three of the seven EcoR1-generated DNA fragments could then be ordered by treating the two cloned HindIII DNA fragments with EcoR1. This led the assignment of the two HindIII restriction sites. The various restriction DNA fragments were hybridized directly from the gel utilizing 32P-labeled 5 S, 5.8 S, 18 S, and 25 S rRNA. Identification of the various DNA restriction segments then led to the final ordering of the DNA fragments. The gene coding for the 5 S RNA is adjacent to the gene coding for the 35 S precursor rRNA. These two groups of genes thus occur as a cluster in the following sequence: [5 S-spacer]-[spacer-18 S-5.8 S-25 S-spacer]-[spacer-5 S]. The actual map of the DNA restriction fragments is presented.     

A Method for Molecular Analysis of Catalase Gene Diversity in Seawater

Catalase plays an important role in the metabolism of marine bacteria and has potential impact on the marine environment. Four PCR primers were designed to amplify the catalase gene fragments in marine bacteria by applying metagenomic DNA from Yellow Sea surface water as the template. Of the four reproducible target PCR products, the longest one with 900 bp were chosen for catalase gene library construction by the T-vector and the white Escherichia coli colonies in the library was screened through restriction-digesting the reamplified insert fragments by the selected restriction endonuclease MboI, and then the bands of the resulting products were displayed in the agarose gel by electrophoresis. The unique restriction fragment length polymorphism (RFLP) pattern was selected and the corresponding catalase gene fragments were sequenced, which verified that every unique RFLP pattern represented one type of catalase. This PCR–RFLP method above was established to investigate the bacterial catalase diversity in seawater.     

Novel restriction fragment length polymorphism of the growth hormone gene in inbred rats

A novel restriction fragment length polymorphism in inbred rats was detected by Southern blot analysis with rat growth hormone cDNA as a probe. Four alleles, characterized by PstI fragments of 1.2, 1.1, 0.9, and 0.7 kb, respectively, were detected in 27 strains examined. The same distribution of polymorphisms was observed on digestion of DNAs of these strains with three other enzymes, PvuII, HindIII, and BamHI. Moreover, the same differences in length of allelic restriction fragments were obtained with these restriction enzymes as with PstI. These findings suggested that the polymorphism was caused by insertion or deletion of variable DNA segments in the second intron of the growth hormone gene. Linkage analyses using backcross progeny provided no evidence for close linkage between the restriction fragment length polymorphism locus and 10 other loci examined.     

Association of transformation of xenosomes from nonkiller to killer with extrachromosomal DNA.

Extrachromosomal DNA in the form of covalently closed circular DNA molecules was isolated from killer and nonkiller xenosomes, bacterial endosymbionts of the marine protozoan Parauronema acutum. Restriction endonuclease digests of these molecules derived from 12 isolates revealed consistent, readily identifiable, differences in the pattern of fragments of the killer as compared with those present in the nonkiller. Transformation of the nonkiller to killer by infection is also accompanied by a change from the nonkiller to killer pattern. Based on analysis of fragments resulting from restriction endonuclease digests, two circular duplex DNA molecules, each 63 kilobase pairs (kbp) in length, were identified in the 263-20 nonkiller stock and mapped. The maps revealed that each possesses a single BamHI site and multiple BglI, BstIIE, PstI, and SalI sites. A distinguishing feature of these maps is that the two molecules share a region about 17 kbp in length in which multiple restriction sites are in register with each other. Allowing for a 0.5-kbp insertion or deletion and the introduction or removal of only a few restriction sites, an additional stretch extending approximately 31 kbp beyond this sequence could also be considered to be homologous. The structure of the killer plasmid appears to be more complex, and we have been unable, as yet, to construct physical maps for this DNA. We postulate that the killer plasmid DNA is composed of three, perhaps four, circular 63-kbp duplexes, at least one which contains a single BamHI site and another which contains two BamHI sites. The remaining molecules may represent copies of either or both of the other two, modified to contain additional restriction sites. Transformation from the nonkiller to the killer is visualized as the insertion of restriction sites at various points along parent nonkiller plasmid DNA molecules. The mechanism by which these sites are introduced is unknown.     

Restriction endonuclease map of Euglena gracilis chloroplast DNA.

A physical map of the Euglena gracilis chloroplast genome has been constructed, based on cleavage sites of Euglena gracilis chloroplast DNA treated with bacterial restriction endonucleases. Covalently close, circular chloroplast DNA is cleaved by restriction endonuclease SalI into three fragments and by restriction endonuclease BamHI into six fragments. These nine cleavage sites have been ordered by fragment molecular weight analysis, double digestions, partial digestions, and by digestion studies of isolated DNA fragments. A fragment pattern of the products of EcoRI restriction endonuclease digestion of Euglena chloroplast DNA is also described. One of these fragments has been located on the cleavage site map.     

Identification of bovine K-casein genotypes at the DNA level

By using a bovine kappa-Cn cDNA as probe and the PstI endonuclease we demonstrate that the DNA restriction patterns of kappa-Cn AA and kappa-Cn BB cows are different. Besides two invariant fragments (about 6.8kb and 1.1kb) the former shows two fragments of about 4.3 kb and 0.3 kb and the latter one fragment of about 4.6 kb. kappa-Cn AB cows show intermediate pattern. Therefore, it is possible to determine the bovine kappa-Cn genotypes even in absence of gene product.     

Patterns of integration of viral dna sequences in the genomes of adenovirus type 12-transformed hamster cells

The patterns of integration of the viral genome have been analyzed in four hamster cell lines transformed by adenovirus type 12 (Ad12). It has previously been shown that in each of the cell lines HA12/7, T637, A2497-2 and A2497-3, the viral genome persists in multiple copies, and that different parts of the viral DNA are represented non-stoichiometrically (Fanning and Doerfler, 1976). All four cell lines are oncogenic when injected into hamsters.The DNA from each of the cell lines was extracted and cleaved in different experiments with restriction endonucleases Bam HI, Bgl II, Eco RI, Hind III, Hpa II or Sma I. The DNA fragments were separated on 1% agarose slab gels and transferred to nitrocellulose filters by the Southern technique. Ad12 DNA sequences were detected by hybridization to Ad12 DNA, which was ³²P-labeled by nick translation, and by subsequent autoradiography. In some experiments, the ³²P-labeled Eco RI restriction endonuclease fragments of Ad12 DNA were used to investigate the distribution of specific segments of the viral genome in the cellular DNA.For each cell line, a distinct and specific pattern of integrated viral DNA sequences is observed for each of the restriction endonucleases used. Moreover, viral sequences complementary to the isolated Eco RI restriction endonuclease fragments are also distributed in patterns specific for each cell line. There are striking differences in integration patterns among the four different lines; there are also similarities. Because the organization of cellular genes in virus-transformed as compared to normal cells has not yet been determined, conclusions about the existence or absence of specific integration sites for adenovirus DNA appear premature. Analysis of the integration patterns of Ad12 DNA in the four hamster lines investigated reveals that some of the viral DNA molecules are fragmented prior to or during integration. Analysis with specific restriction endonuclease fragments demonstrates that the Eco RI B, D and E fragments, comprising a contiguous segment from 0.17–0.62 fractional length units of the viral DNA, remain intact during integration in a portion of the viral DNA molecules. Although each cell line carries multiple copies of Ad12 DNA, the viral DNA sequences are concentrated in a small number of distinct size classes of fragments. This finding is compatible with, but does not prove, the notion that at least a portion of the viral DNA sequences is integrated into repetitive sequences, or else that the integrated viral sequences have been amplified after integration.In the three cell lines which were tested, the integration pattern is stable over many generations, with continuous passage-twice weekly-of cells for 6–7 months. In the three cell lines which were examined, the integration pattern is identical in a number of randomly isolated clones. Hence it can be concluded that the patterns of integration are identical among all cells in a population of a given line of transformed cells.