Cloning of a cDNA encoding the smallest neurofilament protein from the rat

We have cloned a cDNA coding for the smallest rat neurofilament protein. The cDNA is 861 nucleotides long coding for 287 amino acids from the internal alpha-helical region and the carboxy-terminal tail domain of the neurofilament protein. Comparison of the porcine, mouse and rat neurofilament protein sequences shows that the protein is highly conserved (greater than 93% identity). Blot analysis indicates that the cDNA is derived from a single neurofilament gene that codes for two different poly(A)+ mRNA species.     

Sequence analysis of the large and small subunits of human ribonucleotide reductase.

We have isolated and sequenced overlapping cDNA clones from a breast carcinoma cDNA library containing the entire coding region of both the R1 and R2 subunits of the human ribonucleotide reductase gene. The coding region of the human R1 subunit comprises 2376 nucleotides and predicts a polypeptide of 792 amino acids (calculated molecular mass 90,081). The sequence of this subunit is almost identical to the equivalent mouse ribonucleotide reductase subunit with 97.7% homology between the mouse and human R1 subunit amino acid sequences. The coding region of the human R2 subunit of ribonucleotide reductase comprises 1170 nucleotides and predicts a polypeptide of 389 amino acids (calculated molecular mass 44,883), which is one amino acid shorter than the equivalent mouse subunit. The human and mouse R2 subunits display considerable homology in their carboxy-terminal amino acid sequences, with 96.3% homology downstream of amino acid 68 of the human and mouse R2 proteins. However, the amino-terminal portions of these two proteins are more divergent in sequence, with only 69.2% homology in the first 68 amino acids.     

Cloning and characterization of the cDNAs for human and rabbit interleukin-1 precursor.

DNA sequence complementary to the mRNA for rabbit interleukin-1 precursor (preIL-1) has been cloned from the cDNA library constructed using partially purified poly(A)+RNA from induced rabbit alveolar macrophages by mRNA hybridization-translation assay. By using this cDNA as a probe, human IL-1 cDNA was isolated from the cDNA library prepared using poly(A)+RNA from induced HL-60 cells, a human monocyte-like cell line. The amino acid sequences of the human and rabbit preIL-1 deduced from the cDNA sequences reveal their primary structures which consists of 271 and 267 amino acid residues, respectively. The amino acid sequence is 64% conserved between human and rabbit. The difference in number of amino acid residues results from the carboxy-terminal extention of 4 amino acid residues in human preIL-1. Expression of the cloned human cDNA in E. coli yielded biologically active IL-1.     

The mammalian beta-tubulin repertoire: hematopoietic expression of a novel, heterologous beta-tubulin isotype

We describe the structure of a novel and unusually heterologous beta-tubulin isotype (M beta 1) isolated from a mouse bone marrow cDNA library, and a second isotype (M beta 3) isolated from a mouse testis cDNA library. Comparison of M beta 1 and M beta 3 with the completed (M beta 4, M beta 5) or extended (M beta 2) sequence of three previously described beta-tubulin isotypes shows that each includes a distinctive carboxy-terminal region, in addition to multiple amino acid substitutions throughout the polypeptide chain. In every case where a mammalian interspecies comparison can be made, both the carboxy-terminal and internal amino acid substitutions that distinguish one isotype from another are absolutely conserved. We conclude that these characteristic differences are important in determining functional distinctions between different kinds of microtubule. The amino acid homologies between M beta 2, M beta 3, M beta 4, and M beta 5 are in the range of 95-97%; however the homology between M beta 1 and all the other isotypes is very much less (78%). The dramatic divergence in M beta 1 is due to multiple changes that occur throughout the polypeptide chain. The overall level of expression of M beta 1 is low, and is restricted to those tissues (bone marrow, spleen, developing liver and lung) that are active in hematopoiesis in the mouse. We predict that the M beta 1 isotype is functionally specialized for assembly into the mammalian marginal band.     

A cross-species analysis of the cystic fibrosis transmembrane conductance regulator. Potential functional domains and regulatory sites.

To help elucidate the function of the cystic fibrosis transmembrane conductance regulator (CFTR), we have undertaken a cross-species analysis of the DNA sequence which encodes this protein. We have isolated and characterized the cDNA of the bovine homologue of CFTR. The deduced amino acid sequence shows high overall identity with the published sequences from human and mouse, although there is marked variability between the different potential functional domains. The region around human amino acid 508, which is deleted in 70% of cystic fibrosis chromosomes, is highly conserved across species; of the missense cystic fibrosis mutations reported to date, all of the amino acids in the normal human sequence are conserved in the bovine and mouse sequences. A single amino acid encoded by the human cDNA (Ser-434) is missing in the bovine sequence, and there are two amino acids encoded by the bovine sequence which are absent in the human. These all stem from in-frame 3-base omissions within the sequences. In addition to the cow, we amplified the DNA sequences encoding a portion of the R-domain from sheep, monkey, rabbit, and guinea pig. These sequences show relatively low overall sequence identity (63%), but nearly all of the potential protein kinase A and protein kinase C phosphorylation sites are conserved over all of the species examined. Our results suggest functional significance for certain highly conserved residues and putative domains within CFTR.     

A new family of heparin-binding factors: strong conservation of midkine (MK) sequences between the human and the mouse

A retinoic acid responsive gene, MK, specifies for a heparin binding factor termed midkine (MK), which is the initial member of a new protein family involved in regulation of growth and differentiation. A cDNA clone of human MK was isolated from a fetal kidney cDNA library. Human MK mRNA was expressed in PA1 teratocarcinoma cells as well as in the kidney. Sequence analysis of the cDNA clone and of a part of the genomic clone yielded the predicted protein sequence of human MK. Human and mouse MK sequences are highly conserved: 87% of amino acids are identical and all amino acid changes are conservative except for an insertion. Comparison of MK and HB-GAM/pleiotrophin (another member of the family) from various species revealed sequences conserved in the family and those specific for each protein.     

Human Microtubule-Associated Protein 1a (MAP1A) Gene: Genomic Organization, cDNA Sequence, and Developmental- and Tissue-Specific Expression

Microtubule-associated proteins (MAPs) regulate microtubule stability and play critical roles in neuronal development and the balance between neuronal plasticity and rigidity. MAP1a (HGMW-approved symbol MAP1A) stabilizes microtubules in postnatal axons. We describe human MAP1a's genomic organization and deduced cDNA and amino acid sequences. MAP1a is a single-copy gene spanning 10.5 kb. MAP1a coding sequence is contained in five exons. Translation begins in exon 3. Human MAP1a contains 2805 amino acids (predicted molecular weight 306.5 kDa) and is slightly larger than rat MAP1a (2774 amino acids). Like rat and bovine MAP1a, human MAP1a contains conserved tubulin binding motifs in the amino-terminal region. The carboxy-terminal portion contains a conserved pentadecapeptide that is present in the light chain portion of rat and bovine MAP1a/LC2 polyprotein. We show that human MAP1a gene expression occurs almost exclusively in the brain and that there is approximately 10-fold greater gene expression in adult brain compared to fetal brain. Strong, interspecies conservation between human and rat MAP1a cDNA and amino acid sequences indicates important relationships between MAP1a's function and its primary amino acid sequence.     

Molecular cloning of the full-length cDNA encoding mouse neutral ceramidase. A novel but highly conserved gene family of neutral/alkaline ceramidases

We report here the molecular cloning, sequencing, and expression of the gene encoding the mouse neutral ceramidase, which has been proposed to function in sphingolipid signaling. A full-length cDNA encoding the neutral ceramidase was cloned from a cDNA library of mouse liver using the partial amino acid sequences of the purified mouse liver ceramidase. The open reading frame of 2,268 nucleotides encoded a polypeptide of 756 amino acids having nine putative N-glycosylation sites. Northern blot analysis revealed that the mRNA of the ceramidase was expressed widely in mouse tissues, with especially strong signals found in the liver and kidney. The ceramidase activity of lysates of CHOP cells increased more than 900-fold when the cells were transformed with a plasmid containing the cDNA encoding ceramidase. We also cloned the ceramidase homologue from the cDNA library of mouse brain and found that the sequence of the open reading frame, but not the 5'-noncoding region, was identical to that of the liver. Interestingly, phylogenetic analysis of various ceramidases clearly indicated that neutral/alkaline ceramidases form a novel but highly conserved gene family that is evolutionarily different from lysosomal acid ceramidases.     

An mRNA from human brain encodes an isoform of the B subunit of the vacuolar H(+)-ATPase

The B subunit (approximately 60 kDa) of the vacuolar H(+)-ATPase is one of the two major subunits comprising the hydrophilic catalytic complex of the enzyme. Using left and catalytic complex of the enzyme. Using left and right primers which bind two highly conserved sequences of the B subunit, an 836-base pair fragment was amplified from human brain cDNA by the polymerase chain reaction. The amplified fragment was used to probe a Northern blot and to screen a brain cDNA library. A single RNA band, 3.2 kilobases (kb) in length, was detected on Northern blots. A positive cDNA clone containing a 2.5-kb insert was isolated and sequenced. It included a long 3'-untranslated region (greater than 1.2 kb) and was missing a minor portion of the 5'-end of the coding region. The coding region of the brain cDNA sequence was 77% identical at the nucleotide level and 90% identical at the amino acid level to the previously reported sequence for the B subunit of the vacuolar H(+)-ATPase from human kidney (Sudhof, T. C., Fried, V. A., Stone, D. K., Johnston, P. A., and Xie, X.-S. (1989) Proc. Natl. Acad. Sci, U. S. A. 86, 6067-6071). Within the coding region of the brain cDNA, which is 6 amino acid residues shorter at the 3'-end than the kidney sequence, an 11% difference in the GC content was calculated. The 3'-noncoding sequence of the brain cDNA was completely unrelated to that of kidney and was three times longer. We conclude that the B subunit cDNAs from human kidney and brain represent different isoforms. This is the first demonstration of an isoform of a vacuolar H(+)-ATPase subunit.     

Structure and analysis of the bovine atrial natriuretic peptide precursor gene

The isolation and sequence analysis of the gene encoding the bovine atrial natriuretic peptide (ANP) precursor is described. The bovine-ANP coding sequences are located on three exons which are interrupted by two intervening sequences. Comparison of the bovine, human, rat and mouse ANP gene sequences reveals a common organization of introns and exons and a high degree of sequence homology in the 5'-flanking and coding regions. Examination of the pre-proANP amino acid sequence derived from the bovine gene with those from rat, mouse and human, indicates a high degree of sequence homology in both the amino-terminal and biologically-active carboxy-terminal ANP region. The latter region in the bovine sequence resembles its human counterpart except for a carboxy-terminal Arg-Arg dipeptide.     

Mu opioid receptor-like sequences are present throughout vertebrate evolution

The sequence of the mu opioid receptor is highly conserved among human, rat, and mouse. In order to gain insights into the evolution of the mu opioid receptor, polymerase chain reaction (PCR) was used to screen genomic DNA from a number of different species using degenerate oligonucleotides which recognize a highly conserved region. DNA was assayed from representative species of both the protostome and deuterostome branches of the metazoan phylogenetic tree. Mu opioid receptor-like sequences were found in all vertebrate species that were analyzed. These species included bovine, chicken, bullfrog, striped bass, thresher shark, and Pacific hagfish. However, no mu opioid receptor-like sequences were detected from protostomes or from any invertebrates. The PCR results demonstrate that the region of the mu opioid receptor gene between the first intracellular loop and the third transmembrane domain (TM3) has been highly conserved during evolution and that mu opioid receptor-like sequences are present in the earliest stages of vertebrate evolution. Additional opioid receptor-like sequence was obtained from mRNA isolated from Pacific hagfish brain using rapid amplification of cDNA ends (RACE). The sequence of the Pacific hagfish was most homologous with the human mu opioid receptor (72% at the amino acid level between intracellular loop 1 and transmembrane domain 6) although over the same region high homology was also observed with the delta opioid receptor (69%), the kappa receptor (63%), and opioid receptor-like (ORL1) (59%). The hagfish sequence showed low conservation with the mammalian opioid receptors in the first and second extracellular loops but high conservation in the transmembrane and intracellular domains. Received: 5 January 1996 / Accepted: 7 March 1996     

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.