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We have assessed the degree of relatedness of several portions of the Escherichia coli genome to the corresponding portions of the genomes of representative enteric bacteria, using the Southern transfer and hybridization technique (E. Southern, J. Mol. Biol. 98:503-517, 1975). The degree of relatedness varied among the regions examined. Judging both by the relative amounts of deoxyribonucleic acid in the various enteric genomes that are highly homologous and by the conservation of positions of restriction enzyme cleavage sites in these regions, the enteric genomes have diverged to greater extents in some parts of the genomes than in others. Portions of the genomes (including the tnaA and thyA genes, the trp operon, and one other unassigned segment) appear to have evolved in concert with the genome as a whole. By contrast, the lacZ gene and portions of the genome that are homologous to phage lambda vary more widely, perhaps reflecting a separate evolutionary origin for these segments of deoxyribonucleic acid.

Changes in the patterns produced by annealing restriction endonuclease digests of bacterial genomes with probe deoxyribonucleic acids (DNAs) containing small portions of a bacterial genome provide sensitive indicator of the degree of nucleotide sequence relatedness that exists in localized regions of the genomes of closely related bacteria. We have used five probe DNAs to explore the relatedness of parts of the genomes of six laboratory Escherichi coli strains. A range in in the amount of variability in the positions of restriction enzyme cleavage sites in the selected portions of the genomes was found. Portions of the genome that are believed to be inacative were more variable than portions that contained functional genes: the sites in and near regions of homology to phage lambda DNA in the genome showed the greatest variability. These regions probably represent remnants of cryptic prophages. Variability was assessed pairwise among four of the E. coli strains and ranged from 5 to > 25% base pair substitutions in the lambda-related regions. In contrast, the endonuclease cleavage sites in the trp, tna, lac, thy regions, and one other as-yet-unidentified segment of the genome were more highly conserved. It seems likely that these sites lie in genetic locations that are subject to functional constraints.

A group of Escherichia coli isolates from nature were compared with one another and with laboratory strains of E. coli with respect to size distribution of chromosomal restriction endonuclease fragments and differences in nucleotide sequences in selected small portions of the genomes. The estimated frequency of base substitutions in nucleotide sequences in and near the trp operons of 26 of the 28 E. coli strains examined ranged from 0.008 to 0.066. Nucleotide sequences in or near lambda prophage homologs were significantly more variable than the sequences in or near trp, tnaA, and thyA genes. Thus, the lambda-homologous regions may have a significant horizontal component in their evolutionary histories, having undergone genetic exchange, whereas the trp, tnaA, and thyA regions may have solely vertical evolutionary histories. The relatedness of the E. coli strains in the genetic regions studied indicated that laboratory strains are not more closely related to one other than they are to isolates from nature. The isolates from natural populations did not form groups related either by host taxa or by geographical region of isolation.

The chromosomal DNA that lies between the lacI and lacZ genes of Klebsiella pneumoniae constitutes a 196-base pair intercistronic region that contains regulatory sequences for both genes. The probable locations of specific regulatory elements for both lacI and lacZ genes were determined by analogy with the corresponding Escherichia coli sequences. A recombinational event in ancestral DNA evidently has inverted the transcriptional direction of lacI in K. pneumoniae relative to the transcriptional direction of lacI in E. coli. One end of the inversion was located within a 19-base pair sequence in the K. pneumoniae regulatory region. Sequences partially homologous to these 19 base pairs were found in two locations on either side of the E. coli lacI gene. The nucleotide sequence of the lac regulatory region in K. pneumoniae exhibits more than one possibility for folded tertiary structures. The spatial relationships of transcriptional binding sites differ in two possible structures. Associations of regulatory and transcriptional proteins with the DNA might affect conformation of the regulatory sequences and, as a consequence, transcription of the lac genes.

The nucleotide sequences of the Klebsiella pneumoniae lacI and lacZ genes and part of the lacY gene were determined, and these genes were located and oriented relative to one another. The K. pneumoniae lac operon is divergent in that the lacI and lacZ genes are oriented head to head, and complementary strands are transcribed. Besides base substitutions, the lacZ genes of K. pneumoniae and Escherichia coli have suffered short distance shifts of reading frame caused by additions or deletions or both during evolutionary divergence from a common ancestral gene. Relative to corresponding E. coli sequences, the nucleotide sequences of the lacZ and lacY genes are 61 and 67% conserved, and the lacI genes are 49% conserved. A comparison of both nucleotide and amino acid sequences revealed that the K. pneumoniae and E. coli lacI genes and lac repressor proteins each are related to the galR gene and gal repressor of E. coli to about the same extent. In terms of evolutionary relationships, the divergence of the forerunner of the galR gene from an ancestral lac repressor gene preceded separation and differentiation of the K. pneumoniae and E. coli lac repressor genes.

A segment of Escherichia coli DNA that contained a discontinuity of homology with Salmonella typhimurium DNA was isolated. The segment, 1,430 base pairs long, was derived from one end of the lac "loop," a region of about 12 kilobase pairs of E. coli DNA, including the lac operon which has no detectable homology with S. typhimurium DNA (K. Lampel and M. Riley, Mol. Gen. Genet. 186:82-86, 1982). The nucleotide sequence of the 1,430-base-pair segment of DNA was determined. The location of the junction of discontinuity of homology within the segment was established by hybridization experiments. Nucleotide sequences at or near the junction were determined to be similar to sequences that are involved in site-specific inversion in S. typhimurium, E. coli, phage P1, and phage Mu. Similar sequences are also present within the terminal inverted repeat sequences of transposon Tn5 and at the V-D-J joining sequences of eucaryotic immunoglobulin genes. Therefore, the lac operon, together with flanking DNA, may have been inserted into the E. coli chromosome at one time via a site-specific recombination event. Rearrangement events of this kind undoubtedly have played a significant role in the evolutionary divergence of chromosomal DNAs.

We isolated and identified mutant strains of Kluyveromyces lactis that are defective for the Leloir pathway enzymes galactokinase, transferase, and epimerase, and we termed these loci GAL1 , GAL7 , and GAL10 , respectively. Genetic data indicate that these three genes are tightly linked, having an apparent order of GAL7 - GAL10 - GAL1 . This same gene order has been observed in Saccharomyces cerevisiae. Strains harboring gal7 mutations have elevated levels of beta-galactosidase, coded by an unlinked gene, galactokinase, and epimerase activities under uninduced conditions. We investigated the genetic basis of this constitutive gene expression and found no recombinants between the constitutive and Gal- phenotypes among 76 tetrads, suggesting that either GAL7 or a tightly linked gene codes for a regulatory function. This is the second gene that has been shown to specifically coregulate expression of the genes coding for beta-galactosidase and the Leloir pathway enzymes.

An examination of gene expression in diploids may not always be sufficient for determination of the dominant or recessive character of an allele. In Saccharomyces cerevisiae resistance to cryptopleurine has been attributed to a single recessive nuclear gene, cryl, located on chromosome III. We found, contrary to expectations, that resistance to cryptopleurine is not expressed in diploids that are monosomic for chromosome III. Examination of strains of different ploidy on gradient plates shows that the presence of the sensitive allele in a cell does not affect the level of resistance, but rather the level of resistance is directly related to the ratio of resistant alleles to the number of chromosome sets.