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The cloning and nucleotide sequence determination of the Anacystis nidulans R2 phycocyanin (PC) alpha-subunit gene are described. A 3.0-kb PstI fragment of Anacystis nidulans R2 genomic DNA cloned in plasmid pUC8 was found to hybridize with a heptadecameric oligodeoxynucleotide probe. Sequencing using synthetic primers revealed the presence of the PC alpha-subunit gene and the 3' proximal end of the beta-subunit gene. The alpha-gene is separated from the upstream beta-gene by a spacer length of 51 bp. The deduced amino acid (aa) sequence of the alpha-subunit protein is identical, except for 5 aa, to that of A. nidulans 6301 and is highly homologous (77%) to that reported for Agmenellum quadruplicatum PR6. The 16-kDa alpha-subunit protein, detected by immunoadsorption, was fortuitously expressed in Escherichia coli from the lacZ promoter of the cloning vehicle pUC8.

Using the M13 dideoxy sequencing technique, we have established the DNA sequences of colicins E2 and E3 which encompass the receptor-binding and the catalytic domains of each of the nucleases, and their immunity (imm) genes. The imm gene of plasmid ColE2-P9 is 255 bp long and is separated from the end of the col gene by a dinucleotide. This gene pair is arranged similarly in plasmid ColE3-CA38 except that the intergenic space is 9 bp and the E3 imm gene is one codon shorter than its E2 counterpart. Comparisons of the E2 and E3 imm sequences indicate considerable divergence whereas the receptor-binding domains of both colicins are highly conserved. The two nuclease domains appear to share some sequence homology. A possible evolutionary relationship between colicin E3 and other microbial extracellular ribonucleases is also suggested from the sequence alignment analysis.

We have isolated and sequenced the 5' proximal exons and flanking regions of the chicken very low density apolipoprotein II (apo-VLDLII) and serum albumin genes. These genes specify the most abundant mRNA species present in livers of hens or estrogen-treated roosters. Sequencing revealed that the promoter region of the estrogen-dependent apo-VLDLII gene contained at least two potential transcriptional initiation sites, preceded by appropriately positioned "ATA" sequences. One is homologous with the cap site of the ovalbumin gene, and the other exhibits a match of 10 out of 12 nucleotides with the cap site of the serum albumin gene. S1 nuclease mapping indicates that both sites are active in vivo, although the "ovalbumin"-like site is by far the most efficient at all stages of the estrogenic response. The relative efficiencies of these two sites are maintained during in vitro transcription of truncated templates. A third site, that was not anticipated from sequencing data, is active in vivo but inactive in vitro. A conserved 5' flanking element, initially identified during studies on egg white protein genes, is also present upstream from the apo-VLDLII and serum albumin genes. Its removal does not affect initiation site selection in vitro. Regions of the apo-VLDLII and ovalbumin genes extending 100 nucleotides downstream from the "TATA box" contain several striking homologies that suggest a common mode of evolution.

Downstream from its colicin and immunity genes (col. imm), Escherichia coli plasmid ColE3-CA38 contains a 0.81-kb DNA segment, the hic region, which is required for high colicin production. Characterization of derived plasmids, carrying the col-imm operon but varying in the hic region, showed that the latter functions in lacuna production, colicin release, cell death, and lysis. The hic gene expression after induction was shown to be dependent on the col gene promoter. The nucleotide sequence of the 0.81-kb region was determined and the hic gene localized to its imm-distal portion following an open reading frame (ORF) with no known function. There are two overlapping ORFs in that portion of the sequence, one of which was identified as the hic gene by its partial homology to lysis gene H of CloDF13. The 3' half of the hic gene is non-essential and contains a terminator-like DNA sequence. Preceding the gene, there are also inverted repeats which may attenuate its transcription.

The primary structures of the immunity (Imm) and lysis (Lys) proteins, and the C-terminal 205 amino acid residues of colicin E8 were deduced from nucleotide sequencing of the 1,265 bp ClaI-PvuI DNA fragment of plasmid ColE8-J. The gene order is col-imm-lys confirming previous genetic data. A comparison of the colicin E8 peptide sequence with the available colicin E2-P9 sequence shows an identical receptor-binding domain but 20 amino acid replacements and a clustering of synonymous codon usage in the nuclease-active region. Sequence homology of the two colicins indicates that they are descended from a common ancestral gene and that colicin E8, like colicin E2, may also function as a DNA endonuclease. The native ColE8 imm (resident copy) is 258 bp long and is predicted to encode an acidic protein of 9,604 mol. wt. The six amino acid replacements between the resident imm and the previously reported non-resident copy of the ColE8 imm ([E8 imm]) found in the ribonuclease-producing ColE3-CA38 plasmid offer an explanation for the incomplete protection conferred by [E8 Imm] to exogenously added colicin E8. Except for one nucleotide and amino acid change in the putative signal peptide sequence, the ColE8 lys structure is identical to that present in ColE2-P9 and ColE3-CA38.