A dot hybridization method is presented for rapidly determining the relative concentrations of nucleic acids in a mixture, as well as the extent of sequence homology between related RNA or DNA species.

Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure

Human fetal gγ- and Aγ-globin genes: Complete nucleotide sequences suggest that DNA can be exchanged between these duplicated genes

A recombinant phage that carries the cytoplasmic beta-actin gene was isolated from a human DNA library. The nucleotide sequence of this gene was determined. The amino acid sequence deduced from the nucleotide sequence matches perfectly that of beta-actin from human fibroblasts. The gene contains five introns. A large intron was found in the 5' untranslated region six nucleotides upstream from the ATG initiation codon. Four introns were found within the coding region at codons specifying amino acids 41/42, 121/122, 267, and 327/328. In contrast to the human cardiac muscle actin gene, the aorta-type smooth muscle actin gene, and the stomach-type smooth muscle actin gene, the beta-actin gene lacks the codon for cysteine between the ATG initiation codon and the codon for the NH2-terminal amino acid of the mature protein. Hybridization of genomic DNA with DNA fragments derived from intron I in the 5' untranslated region and from intron III strongly suggests the presence of a single beta-actin gene in the human genome. The DNA sequences of the coding region, of the 3' untranslated region, and of the sequence block between the "CCAAT" box and "TATA" box in the 5' flanking DNA of the human beta-actin gene are highly homologous to the corresponding sequences of the rat and chicken beta-actin genes. Unexpectedly, the sequence of intron III of the human beta-actin gene shows considerable homology to that of the rat beta-actin gene.

Molecular structure of the human cytoplasmic beta-actin gene: interspecies homology of sequences in the introns.

Publisher Summary This chapter discusses the plasmids of Escherichia coli ( E. coli ) as cloning vectors. The essence of molecular cloning or recombination in vitro is the joining together in vitro of two or more deoxyribonucleic acid (DNA) fragments. One fragment, called the “vector” or “vehicle,” is capable of replication in some host organism and the other(s), referred to as the “cloned or passenger fragment(s),” can be passively replicated when joined to the vector. Hybrid molecules are then put into a host organism by a process called “transformation,” and the clones that contain various arrangements of the joined fragments are isolated. The chapter discusses the procedures that can be used for the generation of DNA fragments and presents the methods for joining the DNA fragments. The chapter discusses the properties of plasmid cloning vectors of E. coli as well as the techniques that can be used to select from a population of bacterial cells the ones carrying recombinant DNA molecules of interest. The elution of DNA fragments from polyacrylamide gels is also discussed in the chapter.

Plasmids of Escherichia coli as cloning vectors.

The human insulin gene contains two intervening sequences, one is within the region transcribed into the 5'-untranslated segment of the mRNA and the other interrupts the C-peptide encoding region. A comparison of the human with the rat insulin genes indicates potential regulatory regions in the DNA segment preceding the gene and suggests that the ancestral form of the insulin gene had two intervening sequences.

Sequence of the human insulin gene

Cloned β-globin genes of both mouse and rabbit each contain a large and a small intervening sequence (intron) of about equal length at precisely the same positions relative to the coding sequence The homologous introns show some sequence similarity, particularly at the junctions with the coding sequence They most probably arose from a common ancestral sequence and diverged substantially during evolution

Comparison of cloned rabbit and mouse beta-globin genes showing strong evolutionary divergence of two homologous pairs of introns.

Specific excision of the inserted DNA segment from hybrid plasmids constructed by the poly(dA) · poly(dT) method

Cloned β-globin genes of both mouse and rabbit each contain a large and a small intervening sequence (intron) of about equal length at precisely the same positions relative to the coding sequence. The homologous introns show some sequence similarity, particularly at the junctions with the coding sequence. They most likely arose from a common ancestral sequence and diverged substantially during evolution.

Anton Schamböck

Papers

Comparison of cloned rabbit and mouse beta-globin genes showing strong evolutionary divergence of two homologous pairs of introns.

Specific excision of the inserted DNA segment from hybrid plasmids constructed by the poly(dA) · poly(dT) method

THE STRUCTURE OF CLONED RABBIT AND MOUSE β-GLOBIN GENES