Isolation and sequence determination of cDNA encoding the major structural protein of human peripheral myelin.

A full length cDNA of the major structural protein of peripheral myelin (P0 protein) has been isolated from a cDNA library of human fetus spinal cord. The clone is 1948 base pairs (bp) in length and contains a 744 bp open reading frame encoding a polypeptide of 248 residues including 29 signal peptide. The deduced amino acid sequence is highly homologous to P0 protein from other species.     

Isolation and sequence of a cDNA encoding the major structural protein of peripheral myelin

The myelin sheath is a multilayered membrane, unique to the nervous system, which functions as an insulator to increase greatly the velocity of axonal impulse conduction. We have used the techniques of differential screening and hybrid selection to identify a cDNA clone encoding the Schwann cell glycoprotein P0, the major structural protein of the peripheral myelin sheath. The sequence of this protein, deduced from the nucleotide sequence of the cloned cDNA, indicates that P0 is an integral membrane protein containing a single membrane-spanning region, a large hydrophobic extracellular domain, and a smaller basic intracellular domain. The structure of the protein suggests that each of these domains plays an essential role in generating the highly ordered structure of the myelin sheath. Furthermore, we find that the induction of P0 mRNA coincides with the initiation of myelin formation, and we propose a model in which the glycoprotein serves as a molecular guidepost for this process.     

Isolation and sequence determination of cDNA encoding PMP-22 (PAS-II/SR13/Gas-3) of human peripheral myelin.

A full length cDNA of PMP-22 (PAS-II/SR13/Gas-3) of peripheral myelin has been isolated from a cDNA library of human fetus spinal cord. The clone is 1823 base pairs (bp) in length and contains a 480 bp open reading frame encoding a polypeptide of 160 residues. The deduced amino acid sequence is highly homologous to PMP-22 from bovine (PAS-II), rat (SR13) and mouse (Gas-3).     

Characterization of a cloned cDNA encoding rabbit myelin P2 protein

Myelin P2 is a 14,800-Da cytosolic protein found in rabbit sciatic nerves. It belongs to a family of fatty acid binding proteins and shows a 72% amino acid sequence similarity to aP2/422, the adipocyte lipid binding protein, a 58% sequence similarity to rat heart fatty acid binding protein, and a 40% sequence similarity to cellular retinoic acid binding protein. In order to isolate cDNA clones representing P2, a cDNA library was constructed from poly(A+) RNA isolated from sciatic nerves of 10-day-old rabbit pups. By use of a mixed synthetic oligonucleotide probe based on the rabbit P2 amino sequence, 12 cDNA clones were selected from about 25,000 recombinants. Four of these were further characterized. They contained an open reading frame, which when translated, agreed at 128 out of 131 residues with the known rabbit P2 amino acid sequence. These cDNAs recognize a 1.9-kilobase mRNA present in sciatic nerve, spinal cord, and brain, but not present in liver or heart. The levels of P2 mRNA parallel myelin formation in sciatic nerve and spinal cord with maximal amounts being detected at about 15 postnatal days. This initial study will allow characterization of the P2 gene and its regulation, as well as further studies into the role of P2, the first metabolically active myelin-specific protein to be characterized at the genetic level.     

Isolation and analysis of the gene encoding peripheral myelin protein zero

We have isolated the gene encoding the Schwann cell glycoprotein P0, the major structural protein of the peripheral myelin sheath. In rats and mice, this gene is split into six exons distributed over 7 kb of DNA. The segregation of these exons is consistent with the functional segregation of the P0 protein into extracellular, membrane-spanning, and cytoplasmic domains. We find that the P0 extracellular domain is similar in structure to a single immunoglobulin variable region domain. In contrast to prototypical immunoglobulin domains, however, this P0 domain is encoded by two exons, the partitioning of which provides genetic evidence for the evolution of immunoglobulin-related domains from an ancestral half-domain. We also describe procedures for transfection of cultures of nontransformed rat Schwann cells and use these procedures to show that the Schwann cell-specific expression of the P0 gene is controlled by cis-acting elements localized upstream of exon I.     

Isolation of a cDNA encoding the human GM2 activator protein

The GM2 activator protein is a glycolipid-binding protein required for the lysosomal degradation of ganglioside GM2. A human fibroblast cDNA library was screened with mixtures of oligonucleotide probes corresponding to four different areas of the amino acid sequence. A putative clone (821 bp) which gave positive signals to all four probe mixtures was purified and sequenced. The sequence was colinear with the sequence of 160 amino acids of the mature GM2 activator protein. Availability of the cDNA clone should facilitate investigation into function of the GM2 activator protein and also into genetic abnormalities underlying GM2 gangliosidosis AB variant.     

Isolation and characterization of a cDNA encoding the zebra finch myelin proteolipid protein

A cDNA was isolated from a zebra finch telencephalon cDNA library that encodes the myelin proteolipid protein. The clone was 2874 nucleotides long containing an open reading frame of 831 nucleotides that encoded a 277 amino acid myelin proteolipid protein. The 5-and 3 untranslated regions were 112 and 1931 nucleotides, respectively. In Northern blots the clone hybridized to 3 bands of 3.5, 2.4 and 1.5 Kb in mouse brain RNA, but to only a single band of 3.0 kb in zebra finch brain RNA, suggesting the lack of alternative polyadenylation sites within the 3 untranslated region of the zebra finch PLP mRNAs. There was a small degree of homology between the zebra finch and chicken PLP 5 untranslated regions, but relatively little homology of the 5 untranslated regions of the zebra finch PLP cDNA clone with the homologous regions of PLP cDNAs of many mammalian species. Except for a small stretch of considerable homology, there was little overall homology with the 3 untranslated regions of mammalian PLP mRNAs. Approximately 10% (i.e. 28) of the amino acids in the zebra finch PLP differed from mammalian PLP, with most of these changes located within exon 3. There were 16 amino acid changes between zebra finch and chicken, suggesting that greater sequence variation in PLP structure is tolerated among avian species than among mammalian species.Abbreviations DM20 25 kDa proteolipid protein in myelin - PLP classic 30 kDa myelin proteolipid protein Special issue dedicated to Dr. Marjorie B. Lees.     

Isolation and sequence of cDNA clones encoding rat phosphatidylinositol transfer protein

Phosphatidylinositol (PtdIns) transfer protein is a cytosolic protein that catalyzes the transfer of PtdIns between membranes. It is expressed in organisms from yeast to man, and activity has been found in all animal tissues examined. Using antibodies prepared against bovine brain PtdIns transfer protein, lambda gt11 rat brain cDNA libraries were screened and several clones isolated. DNA sequence analysis showed that the cDNAs encoded a polypeptide of 271 amino acids with a mass of 31,911 Da. Comparison of the deduced amino acid sequence with N-terminal sequence data obtained for the intact purified bovine brain protein and rat lung phospholipid transfer protein verified that the cDNAs were PtdIns transfer protein clones. The predicted protein shows no significant sequence similarity to other known (phospholipid)-binding proteins. DNA blot hybridization suggests that the rat genome may contain more than one gene encoding PtdIns transfer protein. RNA blot hybridization reveals that the PtdIns transfer protein gene is expressed at low levels in a wide variety of rat tissues; all tissues examined showed a major mRNA component of 1.9 kilobases and a minor component of 3.4 kilobases. The isolation of clones encoding rat PtdIns transfer protein will greatly facilitate studies of the structure and function of PtdIns transfer proteins and their role in lipid metabolism.     

Isolation and expression of a full-length cDNA encoding the human GM2 activator protein

We report the construction of a cDNA clone encoding a functional GM2-activator protein. The sequence of the complete 5' end of the coding region was determined by direct nucleotide sequencing of a fragment generated by multiple RACE PCR procedures from Hela cell cDNA. Specific oligonucleotides were synthesized from these data which allowed us to produce a PCR fragment that contained the complete coding sequence of the protein. This was then cloned into a mammalian expression vector. The ability of purified hexosaminidase A (beta-N-acetylhexosaminidase, EC 3.2.1.52) to hydrolyse labeled GM2 ganglioside was enhanced 10-fold more by the addition in the assay mix of lysate from transfected COS-1 cells than by the addition of identical amounts of lysate from mock transfected cells. Direct sequencing of PCR fragments from two sources also identified three polymorphisms.     

Isolation and sequence of a cDNA encoding mouse IMP dehydrogenase.

Inosinic acid (IMP) dehydrogenase (IMPD) catalyzes the conversion of IMP to XMP as the first committed step in GMP biosynthesis de novo. We have isolated a cDNA containing the complete coding region of mouse IMPD by its ability to complement a bacterial mutant lacking IMPD activity. Two independent cDNA clones were isolated by complementation, of which the longest was 1.7 kb in length. Northern analyses, using the IMPD cDNA as a probe, indicated that mature IMPD mRNA was a single species approx. 2.0 kb in size. Mouse IMPD is almost identical to Chinese hamster and human IMPDs and is highly conserved between Escherichia coli and mouse, with a direct amino acid (aa) identity of 39%, which increases to 60% if conserved aa are considered. The leader region of our longest cDNA clone is G + C-rich and contains two tandem copies of a G + C-rich direct repeat.     

Cloning and sequence determination of a cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase.

A partial cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase (ACMPK) was isolated from a lambda ZAP expression library by immunoselection using monospecific polyclonal antibodies to the enzyme. The sequence of both strands of the cDNA (CMKa) was determined. The deduced amino acid (aa) sequence contained all eleven consensus domains found in serine/threonine protein kinases [Hanks et al., Science 241 (1988) 42-52], as well as a putative calmodulin-binding domain. The cDNA contained an intron, lacked an in-frame start codon, and was not polyadenylated. A full-length copy of CMKa was subsequently isolated from a lambda gt10 library of A. nidulans cDNA using a restriction fragment of the first clone as a probe. It contained an in-frame start codon, an open reading frame (ORF) of 1242 bp and was polyadenylated. The ORF encoded a protein of 414 aa residues with an M(r) of 46,895 and an isoelectric point pI = 6.4. These values are in good agreement with that observed for the native enzyme [Bartelt et al., Proc. Natl. Acad. Sci. USA 85 (1988) 3279-3283]. When aligned to optimize homology, 29% of the predicted aa sequence of ACMPK is identical to that of the alpha-subunit of rat brain calmodulin-dependent protein kinase II. ACMPK shares 40 and 44% identity in aa sequence with YCMK1 and YCMK2, respectively, two Ca2+/calmodulin-dependent protein kinases recently cloned from Saccharomyces cerevisiae [Pausch et al., EMBO J. 10 (1991) 1511-1522]. Results of Southern analysis of restriction digests of genomic DNA indicate that ACMPK is encoded by a single-copy gene.     

Isolation and sequence analysis of a cDNA encoding rat liver alpha-L-fucosidase.

cDNA clones for alpha-L-fucosidase were isolated from a rat liver lambda gt11 expression library by using both monospecific polyclonal antibodies against the affinity-purified enzyme and biotinylated rat liver fucosidase cDNA sequences as probes. The largest clone, lambda FC9, contained a 1522 bp full-length cDNA insert (FC9) that encoded the 434-amino acid-residue subunit (Mr 50439) of rat liver alpha-L-fucosidase. A putative signal peptide 28 amino acid residues in length preceded the sequence for the mature protein. In addition, FC9 specified for 11 nucleotide residues of 5' untranslated sequence, 78 nucleotide residues of 3' untranslated sequence and a poly(A) tail. The deduced amino acid sequence from FC9 in conjunction with the experimentally determined N-terminus of the mature enzyme suggested that rat liver fucosidase did not contain a pro-segment. However, there was the possibility of limited N-terminal processing (one to five amino acid residues) having occurred after removal of the predicted signal peptide. Amino acid sequences deduced from FC9 were co-linear with amino acid sequences measured at the N-terminus of purified fucosidase and on two of its CNBr-cleavage peptides. An unusual aspect of rat liver alpha-L-fucosidase protein structure obtained from the FC9 data was its high content of tryptophan (6%). The coding sequence from FC9 showed 82% sequence identity with that from a previously reported incomplete human fucosidase sequence [O'Brien, Willems, Fukushima, de Wet, Darby, DiCioccio, Fowler & Shows, (1987) Enzyme 38, 45-53].