Complete nucleotide sequence of a methylcholanthrene-inducible cytochrome P-450 (P-450d) gene in the rat

The rat cytochrome P-450d gene which is inducibly expressed by the administration of 3-methylcholanthrene (MC) has been cloned and analyzed for the complete nucleotide sequence. The gene is 6.9 kilobases long and is separated into 7 exons by 6 introns. The insertion sites of the introns in this gene are well-conserved as compared with those of another MC-inducible cytochrome P-450c gene, but are completely different from those of a phenobarbital-inducible cytochrome P-450e gene. The overall homologies in the coding nucleotide and deduced amino acid sequences were 75% and 68% between the two MC-inducible cytochrome P-450 genes, respectively. The similarity of the gene organization between cytochrome P-450d and P-450c as well as their homology in the deduced amino acid and the nucleotide sequences suggests that these two genes of MC-inducible cytochromes P-450 constitute a different subfamily than those of the phenobarbital-inducible one in the cytochrome P-450 gene family. In contrast with the notable sequence homology in the coding region of the two MC-inducible cytochromes P-450, all the introns and the 5'- and 3'-flanking regions of the two genes showed virtually no sequence homology between them except for several short DNA segments that are located in the promoter region and the first intron. The nucleotide sequences and the locations of these conserved short DNA segments in the two genes suggest that they may affect the expression of the genes. Middle repetitive sequence reported as ID or identifier sequence were found in and in the vicinity of the cytochrome P-450d gene.     

Characterization of a mouse somatic cytochrome c gene and three cytochrome c pseudogenes.

Mouse contains two functional, but differentially expressed, cytochrome c genes. One of these genes is expressed in all somatic tissues so far examined. The other gene is expressed only in testis and is assumed to be spermatogenesis-specific. The nucleotide sequence of four mouse cytochrome c-like genes has been determined. One of these genes (MC1) contains an intron and encodes a polypeptide sequence identical to the published mouse somatic cytochrome c amino acid sequence. The other three genes can not properly encode a mouse cytochrome c protein and appear to be pseudogenes which have arisen via an insertion into the mouse genome of a cDNA copy of a cytochrome c mRNA molecule.     

Isolation and structure of a rat cytochrome c gene

We screened a Charon 4A-rat genomic library using the cloned iso-1 cytochrome c gene from Saccharomyces cerevisiae as a specific hybridization probe. Eight different recombinant phages homologous to a coding region subfragment of the yeast gene were isolated. Nucleotide sequence analysis of a 0.96-kilobase portion of one of these established the existence of a gene coding for a cytochrome c identical in amino acid sequence with that of mouse. The rat polypeptide chain sequence had not previously been determined. In contrast to the yeast iso-1 and iso-2 cytochrome c genes, neither of which have introns, the rat gene contains a single 105-base pair intervening sequence interrupting glycine codon 56. The overall nucleotide sequence homology between cytochrome c genes of yeast and rat is about 62%, with areas of greater homology coinciding with four regions of functionally constrained amino acid sequences. Two of these regions displayed 85-90% DNA sequence homology, including the longest consecutive homologous stretch of 14 nucleotides, corresponding to amino acids 47-52 of the rat protein. Somewhat less homology was observed in the DNA-specifying amino acids 70-80, which are invariant residues in most known cytochrome c molecules. Thermal dissociation of the yeast probe from the homologous rat DNA was at about 58 degrees C in 0.39 M Na+. These results establish that cytochrome c genes may be isolated by interspecies hybridization between widely divergent organisms.     

Molecular evolution of phosphoenolpyruvate carboxylase for C4 photosynthesis in maize: comparison of its cDNA sequence with a newly isolated cDNA encoding an isozyme involved in the anaplerotic function.

Molecular events associated with the evolution of an enzyme for C4 photosynthesis were investigated. In maize, at least three isozymes of phosphoenolpyruvate carboxylase [EC 4.1.1.31] are known: the C4-form, the C2-form and the root-form, being named according to their physiological roles and pattern of tissue distribution [Ting, I.P. & Osmond, C.B. (1973) Plant Physiol. 51, 448-453]. A cDNA clone which presumably encodes the C3-form isozyme was newly isolated and analyzed. Comparison of the sequences of the C3-form and C4-form isozymes revealed that (i) the homologies in the nucleotide and deduced amino acid sequences were 71 and 77%, respectively, and (ii) the gene for the C4-form isozyme evolved under strong G/C pressure. The genes for these isozymes were found to be located apart on different chromosomes. A phylogenetic tree was constructed using 8 amino acid sequences of phosphoenolpyruvate carboxylases from various sources. The topology of the tree indicated that, at least in monocots such as maize and sorghum, the genes for the C4-form and C3-form isozymes diverged from their common ancestral gene earlier than the monocot-dicot divergence (about 2 x 10(8) yr ago), though the divergence of maize (C4 plant) from wheat (C3 plant) is supposed to have occurred much later (6 x 10(7) yr ago).     

The complete amino acid sequence of the human aldolase C isozyme derived from genomic clones

The complete protein sequence of the human aldolase C isozyme has been determined from recombinant genomic clones. A genomic fragment of 6673 base pairs was isolated and the DNA sequence determined. Aldolase protein sequences, being highly conserved, allowed the derivation of the sequence of this isozyme by comparison of open reading frames in the genomic DNA to the protein sequence of other human aldolase enzymes. The protein sequence of the third aldolase isozyme found in vertebrates, aldolase C, completes the primary structural determination for this family of isozymes. Overall, the aldolase C isozyme shared 81% amino acid homology with aldolase A and 70% homology with aldolase B. The comparisons with other aldolase isozymes revealed several aldolase C-specific residues which could be involved in its function in the brain. The data indicated that the gene structure of aldolase C is the same as other aldolase genes in birds and mammals, having nine exons separated by eight introns, all in precisely the same positions, only the intron sizes being different. Eight of these exons contain the protein coding region comprised of 363 amino acids. The entire gene is approximately 4 kilobases.     

Evidence for a period of directional selection following gene duplication in a neurally expressed locus of triosephosphate isomerase

A striking correlation between neural expression and high net negative charge in some teleost isozymes led to the interesting, yet untested, suggestion that negative charge represents an adaptation (via natural selection) to the neural environment. We examine the evolution of the triosephosphate isomerase (TPI) gene family in fishes for periods of positive selection. Teleost fish express two TPI proteins, including a generally expressed, neutrally charged isozyme and a neurally expressed, negatively charged isozyme; more primitive fish express only a single, generally expressed TPI isozyme. The TPI gene phylogeny constructed from sequences isolated from two teleosts, a single acipenseriform, and other TPI sequences from the databases, supports a single gene duplication event early in the evolution of bony fishes. Comparisons between inferred ancestral TPI sequences indicate that the neural TPI isozyme evolved through a period of positive selection resulting in the biased accumulation of negatively charged amino acids. Further, the number of nucleotide changes required for the observed amino acid substitutions suggests that selection acted on the overall charge of the protein and not on specific key amino acids.     

Structure and expression of rodent genes encoding the testis-specific cytochrome c. Differences in gene structure and evolution between somatic and testicular variants

Mammalian testis contains two forms of cytochrome c, one identical to the form found in somatic tissues and a second that is expressed in a stage-specific manner during spermatogenic differentiation. We have isolated both rat and mouse cDNA clones and the rat gene encoding the testis-specific cytochrome c and determined their DNA sequences. The testicular variant displays a number of notable differences with its somatic counterpart. 1) In contrast to the multipseudogene family derived from mammalian somatic cytochrome c genes, the testis gene is single-copy in genomic DNA with no detectable pseudogenes. 2) The rat testis gene is approximately 7 kilobases (kb) long with three introns totaling nearly 6.5 kb whereas the two introns dividing the 2.1-kb somatic gene occupy only 0.9 kb. Introns differ in position as well as size. 3) The testicular variant has a longer 5'-untranslated leader (230 versus 70 base pairs for the somatic gene) with an upstream open reading frame of 129 base pairs beginning with an AUG in a favorable translational context. 4) A single polyadenylation site in the testicular mRNA (approximately 900 nucleotides) contrasts with the three functionally equivalent sites observed in rat somatic messages. 5) Finally, rat and mouse testis cytochromes c differ at 4 amino acid residues as opposed to the complete sequence identity found in the somatic proteins suggesting a shorter unit evolutionary period for these molecules. These observations are consistent with a duplication of an ancestral cytochrome c gene leading to the emergence of novel structural features and regulatory properties likely associated with the striking tissue specificity of the testicular cytochrome c.     

Human brain glycogen phosphorylase. Cloning, sequence analysis, chromosomal mapping, tissue expression, and comparison with the human liver and muscle isozymes

We have cloned the cDNA encoding a new isozyme of glycogen phosphorylase (1,4-D-glucan:orthosphosphate D-glucosyltransferase, EC 2.4.1.1) from a cDNA library prepared from a human brain astrocytoma cell line. Blot-hybridization analysis reveals that this message is preferentially expressed in human brain, but is also found at a low level in human fetal liver and adult liver and muscle tissues. Although previous studies have suggested that the major isozyme of phosphorylase found in all fetal tissues is the brain type, our data show that the predominant mRNA in fetal liver (24-week gestation) is the adult liver form. The protein sequence deduced from the nucleotide sequence of the brain phosphorylase cDNA is 862 amino acids long compared with 846 and 841 amino acids for the liver and muscle isozymes, respectively; the greater length of brain phosphorylase is entirely due to an extension at the far C-terminal portion of the protein. The muscle and brain isozymes share greater identity with regard to nucleotide and deduced amino acid sequences, codon usage, and nucleotide composition than either do with the liver sequence, suggesting a closer evolutionary relationship between them. Spot blot hybridization of the brain phosphorylase cDNA to laser-sorted human chromosome fractions, and Southern blot analysis of hamster/human hybrid cell line DNA reveals that the exact homolog of the newly cloned cDNA maps to chromosome 20, but that a slightly less homologous gene is found on chromosome 10 as well. The liver and muscle genes have previously been localized to chromosomes 14 and 11, respectively. This suggests that the phosphorylase genes evolved by duplication and translocation of a common ancestral gene, leading to divergence of elements controlling gene expression and of structural features of the phosphorylase proteins that confer tissue-specific functional properties.     

Gene structure and nucleotide sequence for rat cytochrome P-450c

Two clones from rat genomic libraries that contain the entire gene for rat cytochrome P-450c have been isolated. lambda MC4, the first clone isolated from an EcoR1 library, contained a 14-kb insert. A single 5.5-kb EcoR1 fragment from lambda MC4, the EcoR1 A fragment, hybridized to a partial cDNA clone for the 3' end of the cytochrome P-450c mRNA. This fragment was sequenced using the dideoxynucleotide chain termination methodology with recombinant M13 bacteriophage templates. Comparison of this sequence with the complete cDNA sequence of cytochrome P-450MC [Yabusaki et al. (1984) Nucleic. Acids Res. 12, 2929-2938] revealed that the EcoR1 A fragment contained the entire cytochrome P-450c gene with the exception of a 90-bp leader sequence. The gene sequence is in perfect agreement with the cDNA sequence except for two bases in exon 2. A second genomic clone, lambda MC10, which was isolated from a HaeIII library, contains the missing leading sequence as well as 5' regulatory sequences. The entire gene is about 6.1 kb in length with seven exons separated by six introns, all of the intron/exon junctions being defined by GT/AG. Amino- and carboxy-terminal information are contained in exons 2 and 7, respectively. These exons contain the highly conserved DNA sequences that have been observed in other cytochrome P-450 species. Potential regulatory sequences have been located both 5' to the gene as well as within intron I. A comparison of the coding information for cytochrome P-450c with the sequence of murine cytochrome P3-450 and rat cytochrome P-450d revealed a 70% homology in both the DNA and amino acid sequence, suggesting a common ancestral gene. Genomic blot analyses of rat DNA indicated that the 3-methylcholanthrene-inducible family of cytochrome P-450 isozymes is more limited in number compared to the phenobarbital-inducible isozymes. Cross-hybridization studies with human DNA suggest a high degree of conservation between rat cytochrome P-450c and its human homolog although gross structural differences do exist between the two genes.     

Primary structure of the bc1 complex of Rhodopseudomonas capsulata. Nucleotide sequence of the pet operon encoding the Rieske cytochrome b, and cytochrome c1 apoproteins

The nucleotide sequence of the pet operon of Rhodopseudomonas capsulata strain SB1003 has been determined. This operon consists of the petA, petB and petC genes, which encode the Rieske Fe-S protein, cytochrome b and cytochrome c1, respectively, all components of the ubiquinol-cytochrome c2 oxidoreductase. The deduced amino acid sequences of the pet genes show homology to the corresponding proteins from other organisms, and particularly high homologies (over 90% for amino acid and nucleotide sequences) to the previously described fbc operon from a strain previously identified as Rhodopseudomonas spheroides GA. The amino acid sequences of the pet proteins are discussed with reference to the structure and function of the ubiquinol-cytochrome c2 oxidoreductase.     

Evolutionary and structural relationships among the group-specific component, albumin and alpha-fetoprotein.

The group-specific component (Gc) is a plasma protein that binds vitamin D. Recent characterization of human Gc cDNA demonstrated homology with serum albumin and alpha-fetoprotein. This study compares the sequences of the three proteins and demonstrates a strong evolutionary relationship. Albumin, alpha-fetoprotein and Gc evolved from an ancestral gene containing an intragenic triplication. Comparison of the amino acid sequences and patterns of double disulfide bonds suggests that the Gc gene may have diverged from an ancestral gene earlier in evolution than the genes encoding albumin and alpha-fetoprotein. Analysis of the amino acid and nucleotide sequences of the three internal domains of Gc revealed 19-23% amino acid sequence identity and the localization of three homology blocks with 40-44% nucleotide sequence identity. The deduced amino sequence of Gc furnished data for comparing its molecular configuration based on the predicted secondary structure with those predicted for human albumin and alpha-fetoprotein. Utilization of Gc cDNA has also led to the identification of its genomic DNA and detection of a human DNA polymorphism.     

Amino acid sequence of the cytochrome subunit of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis

The complete nucleotide sequence of the gene encoding the cytochrome subunit of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis, and the derived amino acid sequence are presented. The nucleotide sequence of the gene reveals the existence of a typical bacterial signal peptide of 20 amino acid residues which is not found in the mature cytochrome subunit. The gene encoding the cytochrome subunit is preceded by the gene encoding the M subunit. Both genes overlap by 1 bp. The mature cytochrome subunit consists of 336 amino acid residues; 73% of its amino acid sequence was confirmed by protein sequencing work. The mol. wt of the cytochrome subunit including the covalently bound fatty acids and the bound heme groups is 40 500. The internal sequence homology is low, despite the symmetric structure of the cytochrome subunit previously shown by X-ray crystallographic analysis of the intact photosynthetic reaction centre. Sequence homologies to other cytochromes were not found.     

The nucleotide sequence and derived amino acid sequence of cDNA coding for mouse carbonic anhydrase II

The nucleotide sequence of a clone containing mouse carbonic anhydrase (CA) cDNA in pBR322 has been determined. The cloned cDNA contains all of the coding region except for nucleotides specifying the first eight amino acids, and all of the 3' noncoding region, which consists of 700 nucleotides. A cDNA clone was identified which contains an additional 54 bp at the 5' end, so that the complete amino acid sequence of mouse CA could be deduced. This sequence showed a 73-81% homology with other mammalian CA form II isozymes, 56-63% with form I isozymes, and 52-56% with form III isozymes. By examination of the amino acids which are unique and invariant for each isozyme, the mouse amino acid sequence was found to contain 16 of the 23 residues that are unique and invariant to mammalian CA form II isozymes, but only one or no residue for forms I and III, respectively.     

Structure and activation of the human N-ras gene

The normal human N-ras gene has been cloned. In structure and sequence it closely resembles the human H-ras and K-ras genes. The three genes share regions of nucleotide homology and nucleotide divergence within coding sequences and have a common intron/exon structure, indicating that they have evolved from a similarly spliced ancestral gene. The N-ras gene of SK-N-SH neuroblastoma cells has transforming activity, while the normal N-ras gene does not, the result of a single nucleotide change substituting lysine for glutamine in position 61 of the N-ras gene product. From previous studies we conclude that amino acid substitutions in two distinct regions can activate the transforming potential of ras gene products.     

An unstable domain in the vicilin genes of higher plants

The genes for the vicilin seed storage proteins of higher plants code for proteins that fall roughly into two size classes. All the vicilin genes descended from a single ancestral gene, and there is considerable homology among the amino acid sequences of all vicilin proteins except in an NH2-terminal domain that is present only in the vicilins of the large size class. This domain, composed largely of charged residues, has diverged greatly. Its nucleotide composition in all species is high in G + A (sense strand) and the domain contains long polypurine.polypyrimidine tracts. Slippage in replication due to non-B DNA structures (for example, H-DNA) resulting from these tracts is considered to be the cause of the rapid divergence of this domain.     

Ancestral sequence reconstruction in primate mitochondrial DNA: compositional bias and effect on functional inference

Reconstruction of ancestral DNA and amino acid sequences is an important means of inferring information about past evolutionary events. Such reconstructions suggest changes in molecular function and evolutionary processes over the course of evolution and are used to infer adaptation and convergence. Maximum likelihood (ML) is generally thought to provide relatively accurate reconstructed sequences compared to parsimony, but both methods lead to the inference of multiple directional changes in nucleotide frequencies in primate mitochondrial DNA (mtDNA). To better understand this surprising result, as well as to better understand how parsimony and ML differ, we constructed a series of computationally simple "conditional pathway" methods that differed in the number of substitutions allowed per site along each branch, and we also evaluated the entire Bayesian posterior frequency distribution of reconstructed ancestral states. We analyzed primate mitochondrial cytochrome b (Cyt-b) and cytochrome oxidase subunit I (COI) genes and found that ML reconstructs ancestral frequencies that are often more different from tip sequences than are parsimony reconstructions. In contrast, frequency reconstructions based on the posterior ensemble more closely resemble extant nucleotide frequencies. Simulations indicate that these differences in ancestral sequence inference are probably due to deterministic bias caused by high uncertainty in the optimization-based ancestral reconstruction methods (parsimony, ML, Bayesian maximum a posteriori). In contrast, ancestral nucleotide frequencies based on an average of the Bayesian set of credible ancestral sequences are much less biased. The methods involving simpler conditional pathway calculations have slightly reduced likelihood values compared to full likelihood calculations, but they can provide fairly unbiased nucleotide reconstructions and may be useful in more complex phylogenetic analyses than considered here due to their speed and flexibility. To determine whether biased reconstructions using optimization methods might affect inferences of functional properties, ancestral primate mitochondrial tRNA sequences were inferred and helix-forming propensities for conserved pairs were evaluated in silico. For ambiguously reconstructed nucleotides at sites with high base composition variability, ancestral tRNA sequences from Bayesian analyses were more compatible with canonical base pairing than were those inferred by other methods. Thus, nucleotide bias in reconstructed sequences apparently can lead to serious bias and inaccuracies in functional predictions.     

The structural gene for cytochrome c551 from Pseudomonas aeruginosa. The nucleotide sequence shows a location downstream of the nitrite reductase gene

The gene coding for Pseudomonas aeruginosa cytochrome c551 has been cloned and its nucleotide sequence determined. Cytochrome c551 is expressed as a 104 amino acid pre-protein from which a signal peptide of 22 amino acids is cleaved off during the translocation across the cytoplasmic membrane. The gene is located just downstream of the gene coding for nitrite reductase on the Pseudomonas aeruginosa chromosome, suggesting that these genes form an operon.