Physical mapping of complex mRNA populations by "criss-cross" DNA-RNA hybridization

The “criss-cross” hybridization technique, originally developed to construct restriction enzyme-generated linkage maps of DNA was adapted to allow simulataneous size estimates of mRNAs, and their location on such physical maps. The technique consists of blot transferring a ³²P-labeled, gel-fractionated mRNA population to a nitrocellulose filter to which a restriction digest of DNA has previously been blot transferred. The RNA transfer is performed under hybridization conditions and perpendicular to the axis of the DNA pattern. The width of the bands in the DNA and RNA gels are controlled such that the resulting matrix allows every mRNA species to cross every restriction fragment band. Thus whenever an mRNA band intersects a DNA band containing complementary sequences, hybridization can occur, and be detected by autoradiography. Each spot in the resulting pattern has size and map location characteristics determined by the electrophoretic mobility of the mRNA band (relative to ribosomal RNA markers) and the physical coordinates of the DNA fragment on the restriction map. As an example of the technique, at least 12 of the late mRNAs of adenovirus type 2 were located on the SmaI physical map of the 35 kbp genome of the virus. In addition, the transciption orientation of mRNAs was determined by hybridization to separated strands of the BamHI fragments.     

Break-Join Recombination in Phage λ

In phage lambda, when DNA replication is blocked, recombination mediated by the Red pathway occurs only near the double-chain break site, cos, that defines the termini of the virion chromosome. The recombinants initiated by cos contain newly synthesized DNA near cos, in amount corresponding to a few percent of the length of lambda. A restriction enzyme cut delivered to one parent far from cos results in elevated recombination near the restriction site. Recombinants induced by this cut have a similarly small amount of DNA synthesis in these replication-blocked crosses. When restriction cuts are introduced in the presence of normal amounts of all of the DNA replication enzymes, many of the resulting recombinants still enjoy, at most, a small amount of DNA synthesis associated with the exchange event. Thus, these experiments fail to support the previously considered possibility that Red-mediated recombination in lambda proceeds largely through a break-copy pathway.     

Catalytic antisense RNAs produced by incorporating ribozyme cassettes into cDNA.

A simple strategy is described for the generation of catalytic hammerhead-type ribozymes (Rz) that can be used as highly specific endoribonucleases to cleave a particular target RNA. The technique requires that a cloned cDNA fragment is available which encodes at least a part of the target RNA. About 25 different restriction recognition sequences can be utilized to incorporate specifically designed DNA cassettes into the cDNA. Besides some nucleotides which are specific for a certain restriction site, the DNA cassettes contain a sequence corresponding to the catalytic domain of the hammerhead Rz and, optionally, selectable marker genes, that are removable. The resulting recombinant DNA constructs permit the in vitro and in vivo synthesis of novel 'catalytic antisense RNAs' or 'antisense Rz (Az)', which combine two features: (i) they bind like antisense RNA to their specific substrate RNA, and (ii) they cleave their target as hammerhead Rz do. The utility of the strategy to generate Rz was demonstrated experimentally by incorporating a synthetic SalI-specific DNA ribozyme (Rz) cassette into a unique SalI site of a cloned cDNA fragment of plum pox virus (PPV), which is a single-stranded positive sense plant RNA virus, belonging to the group of potyviruses. The resulting Az constructs delivered Az that were directed against the PPV (+) or (-) RNA, respectively, which cleaved their corresponding target RNAs in the expected manner. Besides the synthetic Rz cassette, a comparable SalI-specific Rz cassette, that had been prepared from a specifically designed plasmid and that contained the tet gene inserted into the sequence of the catalytic domain of the Rz, was also incorporated into the SalI site of the PPV cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

A simple procedure for parallel sequence analysis of both strands of 5'-labeled DNA

Ligation of a 5'-labeled DNA restriction fragment results in a circular DNA molecule carrying the two 32Ps at the reformed restriction site. Double digestions of the circular DNA with the original enzyme and a second restriction enzyme cleavage near the labeled site allows direct chemical sequencing of one 5'-labeled DNA strand. Similar double digestions, using an isoschizomer that cleaves differently at the 32P-labeled site, allows direct sequencing of the now 3'-labeled complementary DNA strand. It is possible to directly sequence both strands of cloned DNA inserts by using the above protocol and a multiple cloning site vector that provides the necessary restriction sites. The simultaneous and parallel visualization of both DNA strands eliminates sequence ambiguities. In addition, the labeled circular molecules are particularly useful for single-hit DNA cleavage studies and DNA footprint analysis. As an example, we show here an analysis of the micrococcal nuclease-induced breaks on the two strands of the somatic 5S RNA gene of Xenopus borealis, which suggests that the enzyme may recognize and cleave small AT-containing palindromes along the DNA helix.     

A new method for constructing NotI linking and boundary libraries using a restriction trapper.

We have developed a novel method for constructing NotI linking and boundary libraries using a modified "solid-supported ligation primer" (restriction trapper). The restriction trapper could be used to purify the DNA fragments with a specific restriction enzyme cutting site(s) at their ends. The method uses a ligation and recutting reaction with double-stranded DNA ends of a hairpin-shaped oligolinker which is connected covalently to the surface of the latex beads. Selectivity is based on the specificity of the restriction enzyme for its recognition site, resulting in efficient purification. We applied this technique to the construction of high-quality NotI linking and NotI boundary libraries, which contain almost all the NotI sites of the genome and, in addition, are free of illegitimately ligated clones.     

New cloning vectors and techniques for easy and rapid restriction mapping

We have modified plasmid, phage lambda and cosmid cloning vectors to be of general use for easily and unambiguously determining restriction maps of recombinant DNA molecules. Each vector is constructed so that it contains the rarely found NotI restriction site joined to a short synthetic linker sequence that is followed by a multiple cloning site. DNA cloned into these vectors may be restriction-mapped by either of two methods. In one technique, the cloned DNA is completely digested with NotI, followed by partial digestion with any other restriction enzyme. After electrophoresis and transfer to a nylon membrane, the fragments are hybridized to a labeled probe complementary to the NotI linker. In the second technique, referred to as recession hybridization detection, cloned DNA is digested with NotI and then briefly treated with exonuclease III to recess the 3' ends. After hybridizing a labeled complementary oligodeoxynucleotide to the single-stranded 5' end containing the linker sequence, the DNA is partially digested with another restriction enzyme, electrophoresed and the gel is exposed to x-ray film. With either method the size of each labeled fragment corresponds directly to the distance that a restriction site is located from the NotI linker terminus. Methods for obtaining partial restriction enzyme digests have been devised so that as many as 20 different enzymes may be conveniently mapped on a single gel in little more than a day. The vectors and techniques described may also be adapted to automated or semi-automated devices that read fragment lengths and calculate the resulting restriction map.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Polymorphism in the structure of the yeast mitochondrial tRNA synthesis locus.

Yeast mitochondrial DNA contains a genetic locus, called the tRNA synthesis locus, which codes for information necessary for mitochondrial tRNA biosynthesis. A 9S RNA molecule coded by this locus is thought to be the trans-acting element required for the removal of 5' extensions from tRNA precursors. The DNA coding for this RNA maps to a region of mitochondrial DNA known to contain strain specific restriction site polymorphisms. Comparison of the tRNA synthesis locus in two such strains by sequence analysis demonstrates that the restriction enzyme polymorphisms are due to the deletion/insertion of a 50 base pair GC-rich element in the 5' flanking sequence of the 9S RNA coding region. There are also several differences between the 9S RNA coding region of these two strains which do not interfere with the tRNA synthesis function.     

LoxP-directed cloning: use of Cre recombinase as a universal restriction enzyme.

We have developed a novel way to use the Cre/loxP system for in vitro manipulation of DNA and a technique to clone DNA into circular episomes. The method is fast, reliable, and allowsflexible cloning of DNA fragments into episomes containing a loxP site. We show that a loxP site can serve as a universal target site to clone a DNA fragment digested with any restriction enzyme(s). This technique abolishes the need for compatible restriction sites in cloning vectors and targets by generating custom-designed 5' 3', or blunt ends in the desired orientation and reading frame in the vector Therefore, this method eliminates the limitations encountered when DNA fragments are cloned into vectors with a confined number of cloning sites. The 34-bp loxP sequence assures uniqueness, even when large episomes are manipulated. We present three examples, including the manipulation of a bacterial artificial chromosome. Because DNA manipulation takes place at a loxP site, we refer to this technique as loxP-directed cloning.     

The ribosomal RNA genes on Neurospora crassa mitochondrial DNA are adjacent.

Hybridization of separated 24 S and 17 S ribosomal RNA from Neurospora crassa mitochondrial ribosomes to restriction fragments of mitochondrial DNA leads to the conclusion that the large and small ribosomal RNA are adjacent on the restriction endonuclease cleavage map of the DNA. The distance between the two genes is estimated at 900 basepairs. This result is consistent with the existence of a ribosomal precursor RNA in N. crassa mitochondria and is in contrast to the situation in yeast, where the ribosomal genes are far apart on the mitochondrial DNA. The position of the ribosomal RNA genes on the cleavage map of N. crassa mtDNA provides a start for ordering the Hind III restriction fragments.     

The four ribosomal DNA units of the malaria parasite Plasmodium berghei. Identification, restriction map, and copy number analysis

The four ribosomal RNA genes (rDNA units) of the rodent malaria parasite, Plasmodium berghei, were identified and mapped by restriction enzyme analysis and Southern blot hybridization of genomic DNA. Although the four genes share common characteristics, they appear to be internally different from each other in expanse and sequence. One HindIII site near the 3' end of the coding region for the large rRNA is the only restriction site which we have detected that is conserved in all of the genes. The distance between the conserved HindIII site and the coding region for the small rRNA is at least 1-2 kilobases longer in two of the rDNA units than in a third unit. None of the four rDNA units were linked by restriction mapping where about 150 kilobases of the genome were accounted for. The copy number of two of the rDNA units was determined to be approximately 1 per haploid genome by quantitative analysis of cloned (plasmid) DNA versus genomic DNA digests on Southern blots. The other two genes also appear to be present in 1 copy. Restriction analysis confirms both the low copy number and that these genes are not in an easily recognizable tandem array. The low number of rDNA units requires that new copies of the genome produced during intraerythrocytic growth be active in RNA synthesis soon after their replication.     

Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE).

We have developed a rapid quantitative method (Ms-SNuPE) for assessing methylation differences at specific CpG sites based on bisulfite treatment of DNA followed by single nucleotide primer extension. Genomic DNA was first reacted with sodium bisulfite to convert unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. Amplification of the desired target sequence was then performed using PCR primers specific for bisulfite-converted DNA and the resulting product isolated and used as a template for methylation analysis at the CpG site(s) of interest. This methylation-sensitive technique has several advantages over existing methods used for detection of methylation changes because small amounts of DNA can be analyzed including microdissected pathology sections and it avoids utilization of restriction enzymes for determining the methylation status at CpG sites.     

A novel technique for the rapid preparation of mutant RNAs.

We have developed a novel in vitro mutagenesis technique that allows us to introduce mutations at the level of double-stranded DNA and then transcribe the mutant DNA directly. The technique is useful for those wishing to produce recombinant RNA, particularly if the desired recombinant is the result of an insertion or deletion. It is also useful for the preparation of 3'-truncated RNAs with a defined end. The technique is not dependent on the presence of a convenient restriction site within the target gene, and does not involve construction of a clone or amplification of the mutant DNA within a bacterial host. It is intended as a simple and rapid method for the preparation of roughly 100-200 pmol of mutant RNA, which would be sufficient for obtaining sequence information and assessing the functional consequences of the mutation.