Use of the polymerase chain reaction for homology probing of butyrylcholinesterase from several vertebrates

Genomic blots from man, monkey, cow, sheep, pig, rabbit, dog, rat, mouse, guinea pig, and chicken DNA were hybridized with probes derived from the four exons of the human butyrylcholinesterase gene (BCHE) (Arpagaus, M., Kott, M., Vatsis, K. P., Bartels, C. F., La Du, B. N., and Lockridge, O. (1990) Biochemistry 29, 124-131). Results showed that the BCHE gene was present in a single copy in the genome of all these vertebrates. The polymerase chain reaction was used to amplify genomic DNA from these animals with oligonucleotides derived from the human BCHE coding sequence. The amplified segment contained 423 bp of BCHE sequence including the active site serine of the enzyme (amino acid 198) and a component of the anionic site, aspartate 70. Amplification was successful for monkey, pig, cow, dog, sheep, and rabbit DNA, but unsuccessful for rat, guinea pig, mouse, and chicken DNA. Amplified segments were cloned in M13 and sequenced. The mouse sequence was obtained by sequencing a genomic clone. The highest identity of the human amino acid sequence was found with monkey (100%) and the lowest with mouse (91.5%). The sequence around the active site serine 198, Phe-Gly-Glu-Ser-Ala-Gly-Ala, was conserved in all eight animals as was the anionic site component, aspartate 70. A phylogenetic tree of mammalian butyrylcholinesterases was constructed using the partial BCHE sequences.     

Cloning and sequencing of rhesus monkey pepsinogen A cDNA

The complete nucleotide sequence of Rhesus monkey (Macaca mulatta) pepsinogen A (PGA) cDNA was determined from two partially overlapping cDNA clones, covering the whole coding sequence and part of the flanking sequences. The nucleotide and deduced amino acid sequences were compared to known PGA sequences from other species. The degree of similarity with human PGA appeared to be 96% at the nucleotide sequence level and 94% at the amino acid sequence level. In the coding region the divergence was highest in the activation peptide. The amino acid sequence similarity between Japanese monkey (Macaca fuscata) PGA and Rhesus monkey PGA was shown to be 99%. Using the cDNA as probe in Southern hybridization of EcoRI-digested human and Rhesus monkey genomic DNAs, PGA patterns with inter-individual differences were observed. The hybridization patterns are compatible with the existence of a PGA multigene family in both species.     

A novel cDNA extension procedure. Isolation of chicken fatty acid synthase cDNA clones

We have developed a simple and versatile cDNA extension method using lambda-exonuclease-generated single-stranded DNA as a primer. This plasmid-based cDNA extension method can be used to synthesize unidirectional extensions of the existing cDNA clones or subcloned fragments of the untranslated and exon regions of genomic DNA clones. The method is simple to use and involves no addition of linkers or tailing. We have successfully used this method to isolate 4.6 kilobase pairs of chicken fatty acid synthase cDNA clones, starting from the fragment of a genomic clone coding for the untranslated region of the fatty acid synthase mRNA. About 2.8 kilobase pairs of the cDNA coding for the chicken fatty acid synthase has been sequenced. The sequence has an open reading frame coding for 945 amino acids of the fatty acid synthase. In the sequence, we have identified the enoyl reductase, NADPH binding region, a putative beta-ketoacyl reductase region, and the entire sequences of acyl carrier protein and the thioesterase domains. The arrangement of these partial activities in this sequence confirms the arrangement of these activities as determined through partial proteolytic mapping studies. The amino acid sequence of chicken fatty acid synthase deduced from cDNA sequences shows a high degree of homology with the rat fatty acid synthase sequence, suggesting that these multifunctional proteins are conserved evolutionarily.     

Nucleotide sequence and evolution of a mammalian α-Tubulin messenger RNA

Bacterial clones containing complementary DNA sequences specific for rat brain α-tubulin messenger RNA were constructed. One plasmid, pILαTl, contains >95% of the sequences found in the mRNA: the entire coding sequence as well as extensive 5′ and 3′ untranslated sequences. Comparison of the rat amino acid sequence with the known chicken α-tubulin sequence (Valenzuela et al., 1981) reveals the extraordinary evolutionary stability of α-tubulin protein. The presence of only two interspecies amino acid differences within analogous 411 amino acid sequences predicts that amino acid substitutions in this protein are fixed with a unit evolutionary period (Wilson et al., 1977) of 550 million years (i.e. the time required for a 1% difference to arise within a specific protein in two diverging evolutionary lineages). An analysis of the silent nucleotide differences, permissible because of the degeneracy of the genetic code, demonstrates that these might not occur in a random fashion. The high guanine-cytosine bias in silent codon positions within the chicken α-tubulin sequence, previously noted by Valenzuela et al. (1981), is not conserved within the rat sequence. This decrease in guanine-cytosine bias is accompanied by a selective loss of CpG dinucleotides in the rat sequence.     

A conserved cysteine in molybdenum oxotransferases

The amino acid sequences of peptides derived from rat hepatic sulfite oxidase have been determined by a combination of amino acid analysis and Edman degradation of the purified protein. The data obtained showed the rat liver enzyme contained 3 cysteine residues which was confirmed by thiol modification studies using 4,4'-dithiodipyridine of the native enzyme. Combining these data with that previously published for chicken liver sulfite oxidase (Neame, P. J., and Barber, M. J. (1989) J. Biol. Chem. 264, 20894-20901) indicates that 2 cysteines (Cys186 and Cys430, based upon the numbering for the chicken sequence) are conserved in both chicken and rat liver enzymes with all the cysteine residues being present in the molybdenum-containing domain. Further comparison of the sequences of the molybdenum domains of rat and chicken liver sulfite oxidase with the amino acid sequences published for the molybdenum domains of a variety of assimilatory nitrate reductases suggests that only a single cysteine residue (Cys186) is conserved in all these enzymes, indicating that it may play a role in the binding of Mo-pterin to the protein.     

Molecular cloning and sequencing of cDNAs encoding homologues of human Ku70 and Ku80 autoantigen from Xenopus and their expression in various Xenopus tissues

We isolated cDNA clones encoding Ku70 and Ku80 homologues of Xenopus laevis from a cDNA library prepared from Xenopus oocytes. The nucleotide sequences of these Ku70 and Ku80 homologues have coding sequences of 1833 bp and a 611 aa protein, and 2178 bp and a 726 aa protein, respectively. The amino acid sequences deduced from the open reading frame of the Ku70 and Ku80 cDNA clones were highly homologous to those from Ku genes previously isolated, such as human (ca. 65% and ca. 62% identity, respectively) and mouse (ca. 65% and ca. 60%), and show a certain degree of homology to Drosophila (ca. 27% with Ku70), Caenorhabditis elegans (ca. 20% with Ku80) and Saccharomyces cerevisiae (ca. 23% and ca. 19%). Our detailed comparison of the predicted amino acid sequences among these species revealed the highly conserved octa-peptide LPFXXDIR common to both Xenopus Ku70 and Ku80 homologues in the region showing the high homology throughout the species tested. A Northern analysis using specific cDNA probes showed that Ku poly(A)+ mRNAs are expressed at high levels in Xenopus adult oocyte and testis.     

Molecular cloning and the nucleotide sequence of cDNA for embryonic chicken pepsinogen: phylogenetic relationship with prochymosin

Embryonic chicken pepsinogen is an aspartyl proteinase that is specifically secreted during the embryonic period in the chicken proventriculus (glandular stomach). To learn the phylogeny of this pepsinogen, we isolated a cDNA clone by screening a lambda gt11 library of embryonic proventricular cDNAs with an antiserum to the embryonic chicken pepsinogen. We obtained a 200-base pair cDNA clone which encoded 18 amino acids that had high sequence homology with the carboxyl termini of other pepsinogens. Northern blot analysis revealed that this cDNA clone hybridized to a mRNA of 1,600 bases in the embryonic proventriculus but not to the mRNA in the adult proventriculus. The almost complete nucleotide sequence of embryonic chicken pepsinogen-cDNA was determined by sequencing longer cDNAs obtained by screening the same library with the 200-base pair cDNA and primer extension with a synthetic primer. The cDNA consisted of 1,281 nucleotides and encoded 383 amino acids for prepepsinogen. The predicted amino acid sequence was compared with the sequences of other aspartyl proteinases: pepsinogen A of human, monkey, pig, and chicken, progastricsin of monkey and rat, and bovine prochymosin. The phylogenetic tree constructed for them indicates the possibility that embryonic chicken pepsinogen diverged from prochymosin, after prochymosin and pepsinogen A had diverged from each other.     

A rice (Oryza sativa L.) cDNA encodes a protein sequence homologous to the eukaryotic ribosomal 5S RNA-binding protein

A rice (Oryza sativa L.) cDNA clone coding for the cytoplasmic ribosomal protein L5, which associates with 5 S rRNA for ribosome assembly, was cloned and its nucleotide sequence was determined. The primary structure of rice L5, deduced from the nucleotide sequence, contains 294 amino acids and has intriguing features some of which are also conserved in other eucaryotic homologues. These include: four clusters of basic amino acids, one of which may serve as a nucleolar localization signal; three repeated amino acid sequences; the conservation of glycine residues. This protein was identified as the nuclear-encoded cytoplasmic ribosomal protein L5 of rice by sequence similarity to other eucaryotic ribosomal 5 S RNA-binding proteins of rat, chicken, Xenopus laevis, and Saccharomyces cerevisiae. Rice L5 shares 51 to 62% amino acid sequence identity with the homologues. A group of ribosomal proteins from archaebacteria including Methanococcus vanniellii L18 and Halobacterium cutirubrum L13, which are known to be associated with 5 S rRNA, also related to rice L5 and the other eucaryotic counterparts, suggesting an evolutionary relationship in these ribosomal 5 S RNA-binding proteins.     

Structure, evolution, and tissue-specific synthesis of human apolipoprotein AIV

Apolipoprotein AIV (apoAIV) is a protein of the lipid transport system found associated with chylomicrons, high-density lipoprotein (HDL), and the lipoprotein-free fraction of the plasma. The gene coding for the human apoAIV is closely linked with the genes coding for apolipoproteins AI (apoAI) and CIII (apoCIII). In this paper a nearly full-length apoAIV cDNA clone has been isolated by screening an adult human liver DNA library using a human apoAIV gene probe. In-frame translation of the cDNA sequence in this clone indicated that the human apoAIV consists of 396 amino acid residues including a 20 residue long signal peptide. The coding region of this cDNA sequence contains 15 nucleotide repeats, 11 of which code for amino acid repeats with potentials of forming amphipathic helices. Alignment and comparison of the human and rat apoAIV amino acid sequences indicated a five-residue deletion near the carboxy terminus of the rat protein. This comparison also indicated that these proteins are 61.8% homologous, suggesting that the rate of evolution of apoAIV is 65 accepted point mutations (PAMs) per 100 residues per 100 million years. The rates of evolution of certain amino acid repeats in apoAIV are higher than the rate of evolution of the entire protein. However, the corresponding, computer-generated, secondary structures and hydropathy profiles of these repeats are very similar between the human and rat apoAIV. The relative steady-state levels of apoAIV mRNA in various human and monkey tissues were determined by hybridization blotting analysis of total RNA from these tissues using a human apoAIV cDNA probe.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complete amino acid sequence of rat L-type pyruvate kinase deduced from the cDNA sequence

cDNA clones, containing the entire coding region of rat L-type pyruvate kinase, were isolated and their nucleotide sequences were determined by the dideoxy-chain-termination method. The predicted coding region, which spans 543 amino acids, established the complete amino acid sequence of the L-type isozyme of pyruvate kinase for the first time. The deduced amino acid sequence of the L type has one phosphorylation site in its amino terminus and shows about 68% and 48% homologies with M1-type pyruvate kinase of chicken and yeast pyruvate kinase respectively. Domain A exhibits higher homology than domains B and C. The residues in the active site of the L-type enzyme of rats, lying between domains B and A2, are rather different from those of the M1-type enzyme of chickens, but other residues constituting the active site are identical with those of the chicken M1 type except for one amino acid substitution.     

Nucleotide sequence determination of mouse, chicken and Xenopus laevis rig cDNAs: the rig-encoded protein is extremely conserved during vertebrate evolution

Mouse, chicken and Xenopus laevis homologues to rig (rat insulinoma gene) cDNA were isolated and their nucleotide sequences were determined. Each homologue encoded a 145-amino acid protein; the amino acid sequence remained invariant in the murine and avian genes, and there were only 6 amino acid substitutions in the salientian gene. The evolutionary rate calculated for rig mRNA was sufficiently low to be viewed as evidence that rig is vital to vertebrate species. Southern blot analysis indicated that haploid sets of the mammalian genomes contain several copies of rig or rig-related sequences, whereas there appeared to be only one copy in the amphibian and bird genomes. The possibility that rig belongs to the class of housekeeping genes is discussed.     

Sequence and characterization of cDNA encoding the motilin precursor from chicken, dog, cow and horse. Evidence of mosaic evolution in prepromotilin

Motilin is involved in the regulation of the fasting motility pattern in man and in dog, but may have a different role in other species. Immunoreactive motilin has been demonstrated in several species, but the sequence is mostly unknown. The aim of this study was to isolate and sequence the cDNA encoding the motilin precursor from several mammalian species and from chicken. Total RNA was isolated from the duodenal mucosa of the chicken, dog, cow and horse. In each case single stranded cDNA was synthesized. Motilin cDNA fragments were amplified by PCR, ligated into a plasmid and cloned. Clones which were positive after screening with an appropriate (32)P-labeled probe were sequenced. The 5'- and 3'-ends were determined by the rapid amplification of cDNA ends (RACE) method. Analysis of the cDNAs revealed an open reading frame coding for 115 (chicken and cow), or 117 (dog and horse) amino acids. It consists of a 25 amino acid signal peptide, motilin itself, and a 68 (chicken and cow) or 70 (dog and horse) amino acid motilin associated peptide (MAP). As in all motilin precursors already sequenced (man, monkey, pig and rabbit), an endoproteinase cleavage site is present at Lys(23)-Lys(24). Comparison of all known sequences shows considerable identity in amino acid and nucleotide sequence of the signal peptide and motilin. However, the MAPs differ not only in length but also, more strongly, in amino acid and nucleotide sequence. Our study demonstrates that the N- and C-terminal regions of the motilin precursor have evolved at different rates, which is evidence for 'mosaic evolution'.     

Comparison of the organization and fine structure of a chicken and a Xenopus laevis vitellogenin gene

The structural organization and the coding nucleotide sequence of the Xenopus laevis A2 and the chicken major vitellogenin genes have been compared. Both genes show the same exon-intron organization. However, the degree of homology between the nucleotide and derived amino acid sequences varies extensively along the genes. Several of the 35 exons are quite similar, and a unique cysteine motif in the lipovitellin II domain is conserved between the two genes. In contrast, one internal region is quite divergent. Part of this region encodes phosvitin, which appears to have evolved rapidly by both point mutations and duplications of serines or short other amino acid stretches. On the basis of these observations, we discuss the possible mechanism of evolution of phosvitin in vertebrates.     

Genetic analysis of the cell binding domain region of the chicken fibronectin gene

We have determined the nucleotide sequence of the cell binding domain region of the chicken fibronectin gene and analyzed it evolutionaly. We present here the complete nucleotide sequence of 4.3 kb HindIII/EcoRI segment from the clone lambda FC23 of the chicken fibronectin gene. There were five exons in this segment. When we lined up the amino acid of exons 28, 29 and 31, three alignments, known as the Type III repeat, appeared. Tetrapeptide, -RGDS-, called the cell binding domain, existed in the second repeat, coding exon 30. It was presumed that the Type III repeats were composed of two exons in the chicken gene, the same as in the rat and humans. We found repeatedly appearing amino-acid sequences such as -TIT- (three arrays in these Type III repeats) but also found one of the amino acids substituted in the tripeptide in these Type III repeats (seven arrays). We analyzed these repeats from the point of view of evolution. We used three of the nucleotide sequences (12-18 bp) coding such -TIT- repeats as a unit length for comparing the various homologies after dividing the coding region into 56 segments. The mutual homology of the divided segments to each one of three showed 53% on average. On the other hand, the mutual nucleotide homology of the Type III repeat was 44%. This suggested that the Type III repeat may have been developed by frequent duplication of small gene units.     

42Sp48 in previtellogenic Xenopus oocytes is structurally homologous to EF-1 alpha and may be a stage-specific elongation factor

We have isolated the cDNA for 42Sp48 and EF-1 alpha from mixed stage oocytes and tailbud (stage 22) Xenopus laevis cDNA libraries by use of the cDNA for human elongation factor-1 alpha (EF-1 alpha) as probe. The nucleotide and deduced amino acid sequences of the entire coding region of 42Sp48 and EF-1 alpha cDNA were established. The proposed functional homology of the proteins is reflected in highly conserved amino acid sequences (91% identity), while the large number of silent mutations at the gene level may serve to prevent recombination at their loci. 42Sp48 is apparently encoded by two genes in Xenopus, while no sequences corresponding to 42Sp48 could be found in murine or human genomic DNA. 42Sp48 has been proposed to act as a stage-specific elongation factor in Xenopus. Comparison of the deduced amino acid sequences of 42Sp48 and EF-1 alpha with that of elongation factor Tu from E. coli, for which the three-dimensional structure including that of the GTP binding sites have been determined, supports this hypothesis.     

(AT)n is an interspersed repeat in the Xenopus genome.

We have observed (AT)34 and (AT)23 tracts close to the coding sequences of the Xenopus laevis tadpole alpha T1 and adult beta 1 globin genes, respectively. We show that (AT)n sequences are found as interspersed repeats within the Xenopus globin and histone gene loci. Using (AT)n co-polymer in filter hybridisation experiments we estimate that there are 10(4) (AT)n tracts per haploid Xenopus genome. Hybridisation to genomic blots of DNA from yeast, slime mold, trypanosome, fruit fly, salmon, chicken, rat, human, crab and Xenopus species shows that strictly alternating AT of sufficient length to hybridise appears to be most abundant in Xenopus and crab genomes. We show that the specificity of the co-polymer probe for strictly alternating AT is, however, dependent on the length of the probe. Hybridisation experiments using (TG)n copolymer suggest that this highly conserved repeat is found as clustered repeats in the Xenopus genome in contrast to other eukaryotic genomes so far studied.     

The difference in the carboxy-terminal sequence is responsible for the difference in the activity of chicken and rat liver fructose-2,6-bisphosphatase

The fructose-2,6-bisphosphatase domain of the bifunctional chicken liver enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase shares approximately 95% amino acid sequence homology with that of the rat enzyme. However, these two enzymes are significantly different in their phosphatase activities. In this report, we show that the COOH-terminal 25 amino acids of the two enzymes are responsible for the different enzymatic activities. Although these 25 amino acids are not required for the phosphatase activity, their removal diminishes the differences in the activities between the two enzymes. In addition, two chimeric molecules (one consisting of the catalytic core of the chicken bisphosphatase domain and the rat COOH-terminal 25 amino acids, and the other consisting of most of the intact chicken enzyme and the rat COOH-terminal 25 amino acids) showed the same kinetic properties as the rat enzyme. Furthermore, substitution of the residues Pro456Pro457Ala458 of the chicken enzyme with GluAlaGlu, the corresponding sequence in the rat liver enzyme, yields a chicken enzyme that behaves like the rat enzyme. These results demonstrate that the different bisphosphatase activities of the chicken and rat liver bifunctional enzymes can be attributed to the differences in their COOH-terminal amino acid sequences, particularly the three residues.     

Purification, cDNA cloning, and expression profiles of the cyclobutane pyrimidine dimer photolyase of Xenopus laevis

Photolyase is a light-dependent enzyme that repairs pyrimidine dimers in DNA. Two types of photolyases have been found in frog Xenopus laevis, one for repairing cyclobutane pyrimidine dimers (CPD photolyase) and the other for pyrimidine-pyrimidone (6-4)photoproduct [(6-4)photolyase]. However, little is known about the former type of the Xenopus photolyases. To characterize this enzyme and its expression profiles, we isolated the entire coding region of a putative CPD photolyase cDNA by extending an EST (expressed sequence tag) sequence obtained from the Xenopus database. Nucleotide sequence analysis of the cDNA revealed a protein of 557 amino acids with close similarity to CPD photolyase of rat kangaroo. The identity of this cDNA was further established by the molecular mass (65 kDa) and the partial amino acid sequences of the major CPD photolyase that we purified from Xenopus ovaries. The gene of this enzyme is expressed in various tissues of Xenopus. Even internal organs like heart express relatively high levels of mRNA. A much smaller amount was found in skin, although UV damage is thought to occur most frequently in this tissue. Such expression profiles suggest that CPD photolyase may have roles in addition to the photorepair function.     

Evolution of insulin-like growth factor I (IGF-I): structure and expression of an IGF-I precursor from Xenopus laevis

By means of a cloning strategy employing the polymerase chain reaction, we have isolated and characterized cDNAs for Xenopus laevis insulin-like growth factor I (IGF-I). These cDNAs encode a primary IGF-I translation product of 153 residues that demonstrates considerable amino acid sequence similarity with IGF-IA peptides from other species. Fifty-seven of 70 residues of the mature protein are identical among human, rat, chicken, and Xenopus IGF-I, while less amino acid conservation is found at the COOH-terminus (25/35 identities) or at the NH2-terminus (24/48 identities) of the precursor protein. Despite the lower degree of structural similarity at the NH2-terminus, in vitro studies of IGF-I biosynthesis and proteolytic processing support a conserved function for the atypically long 48 residue NH2-terminal signal sequence in directing the nascent IGF-I peptide through the secretory pathway. The 5'-untranslated region of Xenopus IGF-I mRNA matches the human, rat, and chicken sequences in greater than 90% of 279 nucleotides. IGF-I mRNAs from all four species encode a conserved upstream open reading frame of 14 amino acids starting 240-250 nucleotides 5' to the translation start site, suggesting a possible role for this region in modulating IGF-I gene expression. The X. laevis IGF-I gene is transcribed and processed into three mRNAs of 1.6, 2.1, and 3.0 kilobases in liver, and IGF-I mRNAs can be detected in liver, lung, heart, kidney, and peritoneal fat of adult animals. These studies demonstrate that both the IGF-I protein precursor and potential regulatory regions of IGF-I mRNA have been conserved during vertebrate evolution, and indicate that like several other polypeptide growth factors, IGF-I may be of fundamental importance in regulating specific aspects of growth and development in all vertebrates.