Direct analysis of single nucleotide variation in human DNA and RNA using in situ dot hybridization.

Using oligonucleotide hybridization, single and multiple nucleotide differences between alleles were detected directly in genomic DNA without electrophoretic separation. The DNA was immobilized in depressions in an agarose gel (in situ dots) and hybridized with radiolabeled, allele-specific oligonucleotide probes. An oligonucleotide complementary to a unique sequence region of the human major histocompatibility complex gene HLA-B27 only hybridized with genomic DNA from an HLA-B27-positive individual. Two other oligonucleotides complementary to the normal human beta-globin gene (beta A) and to the sickle cell globin gene (beta S) were synthesized. Using competition hybridization conditions which included the presence of a 10-fold molar excess of unlabeled oligonucleotide complementary to the other beta-globin allele, DNA from individuals homozygous for the normal beta-globin gene (beta A beta A) hybridized to the beta A probe exclusively, whereas DNA from individuals homozygous for the sickle cell globin gene (beta S beta S) hybridized only with the probe for the sickle cell gene. As expected, DNA from heterozygous individuals bound to both probes. Similar results were obtained with total human RNA immobilized in in situ dots. Possible applications of this methodology include genetic disease diagnosis, population carrier screening, HLA "DNA" typing, and DNA and RNA sequence polymorphism analysis.

Enzymatic synthesis of oligoribonucleotides of defined sequence.

A plasmid DNA vector is described that is suitable for cloning synthetic DNA sequences. These cloned synthetic DNA sequences can be transcribed in vitro to produce oligoribonucleotides of defined sequence. Transcription is directed by a promoter based on the consensus sequence for Escherichia coli promoters and uses E. coli RNA polymerase. The vector is useful for cloning oligodeoxyribonucleotides of mixed sequences, the individual sequences being resolved by transformation and colony selection. Oligoribonucleotide synthesis from the vector is highly specific. Application of these sequences in hybridization experiments is demonstrated.

Oligonucleotide-directed mutagenesis using plasmid DNA templates and two primers.

Oligonucleotide-directed mutagenesis using plasmid vectors has been simplified by introducing two changes to the previous method. First, template preparation has been simplified by using the covalently closed circular plasmid DNA directly for mutagenesis, eliminating the need for a wholly or partially single-stranded circular DNA template. Second, two primers are used, eliminating the need for producing the covalently closed molecule during in vitro replication. The advantages of the approach are discussed.

Functional expression of a yeast ochre suppressor tRNA gene in Escherichia coli.

The yeast tRNATyr ochre suppressor SUP6 gene and a derivative of this gene in which the 14-bp intervening sequence has been deleted, SUP6 delta, have been examined for functional expression in Escherichia coli. The SUP6 delta, but not the SUP6, gene codes for a functional transfer RNA which has been shown to suppress both ochre and amber nonsense mutations in E. coli. Although the SUP6 delta fragment is contained within a 750-bp restriction fragment, isolated from Saccharomyces cerevisiae, and contains no encoded CCA, the primary transcript, which originates from an E. coli promoter, approx. 1000 bp upstream, is processed to a mature, functional transfer RNA. The pattern of suppression, i.e., suppression of both ochre and amber mutants, is characteristic of E. coli ochre suppressing tRNAs and is in contrast to the pattern observed in yeast, where only ochre mutations are suppressed. The SUP6 delta encoded tRNA, although coding for tRNATyr in yeast, is not charged solely with tyrosine in E. coli. The functional expression of this mutant eukaryotic transfer RNA gene in E. coli affords a unique opportunity for studies of expression of a gene coding for a stable RNA, in both a prokaryotic and an eukaryotic host.

Isolation of a cDNA clone for the murine transplantation antigen H-2Kb.

A library of cloned cDNAs constructed from the poly(A)+RNA of the murine thymoma cell line EL4 (b haplotype) was screened with a probe encoding a short region of the H-2Kb transplantation antigen. One of the clones isolated, pH202, contains a region that can code for a transplantation antigen with an amino acid sequence 98% homologous to that previously published for H-2Kb. Based on this high degree of homology, pH202 appears to encode the H-2Kb antigen from amino acid 66 through the carboxy terminus, including 386 nucleotides of 3'-untranslated sequence. The amino acid sequence deduced from pH202 suggests that the H-2Kb antigen is actually 2 amino acids longer than previously reported (a total of 348 residues). Four other differences in amino acid assignments are seen. Analysis of the DNA sequences of pH202 and other H-2 clones previously described in the literature suggests that alternative routes of splicing at the 3' end of the coding region are involved in the production of different transplantation antigen mRNAs.

The complete amino acid sequence of the murine transplantation antigen H-2Db as deduced by molecular cloning.

A mouse cDNA library derived from the EL4 cell line (b-haplotype) was screened with a probe containing a small part of the H-2Kb coding region. One of the clones isolated, pH203, encodes a protein whose deduced amino acid sequence is identical with the known sequence of H-2Db in 141 of 141 positions available for comparison. The clone, therefore, is believed to code for the H-2Db transplantation antigen. The cDNA insert of pH203 contains the coding region for residues 82 through the carboxy-terminus of H-2Db, and includes 476 nucleotides of the 3'-untranslated sequence. Comparison between the H-2Db cDNA clone and a previously isolated H-2Kb cDNA clone shows homologies of 83% and 91% at the amino acid and nucleotide levels, respectively. Analysis of DNA sequences at the 3'-coding and untranslated regions suggests that the mRNAs of H-2Kb and H-2Db are spliced differently at their 3'-coding ends.

Oligonucleotide directed mutagenesis of the human beta-globin gene: a general method for producing specific point mutations in cloned DNA.

A nonadecanucleotide has been used both as a site specific mutagen to introduce a T leads to A transversion mutation in the human beta-globin gene cloned in pBR322 as well as a probe to screen transformed colonies for the desired mutant. The specificity of the oligonucleotide as a mutagen and as a hybridization probe provide a general method for producing site specific mutations in DNA cloned in plasmid vectors such as pBR322.

Identification of an H-2Kb-related molecule by molecular cloning.

Based on the published amino-acid sequence of H-2Kb, we synthesized a mixture of eight 16-base long oligodeoxyribonucleotides representing all possible coding sequences for residues 51-56 (Trp-Met-Glu-Gln-Glu-Gly). The hexadecanucleotide mixture was used as a probe to screen recombinant DNA clones constructed from cytoplasmic PolyA+ RNA isolated from the murine thymoma cell line EL4(b haplotype). Of the 30 000 independent clones screened, one clone was found to hybridize with the probe. DNA sequence analysis showed that the cDNA clone was derived from a portion of an H-2Kb -related mRNA. The clone encodes a protein sequence identical with a region of H-2Kb in 42 consecutive residues (50 through 91). The sequence than diverges from the H-2Kb sequence and, after a single Glu codon, a termination codon is encountered. It is possible that this mRNA codes for a small 92 amino-acid protein with a sequence identical (except for a carboxy-terminal Glu residue) with the amino terminus of H-2Kb. It is further speculated that this mRNA is coded for by the H-2Kb gene and differs from the H-2Kb mRNA in the pattern of posttranscriptional splicing.

Directed deletion of a yeast transfer RNA intervening sequence.

Many eukaryotic genes contain intevening sequences, segments of DNA that interrupt the continuity of the gene. They are removed from RNA transcripts of the gene by a process known as splicing. The intervening sequence in a yeast tyrosine transfer RNA (tRNA Tyr) suppressor gene was deleted in order to test its role in the expression of the gene. The altered gene and its parent were introduced into yeast by transformation. Both genes exhibited suppressor function, showing that the intervening sequence is not absolutely essential for the expression of this gene.