Screening of proteins that interact with human thrombopoietin receptor c-Mpl using yeast two-hybrid system

Thrombopoietin (TPO) is the major cytokine involved in platelet production and exerts its effects via the receptor c-Mpl. The yeast two-hybrid system has been used to screen the proteins interacting with c-Mpl. First, the cDNA fragment of c-Mpl intracellular domain was cloned into two-hybrid vector pAS2, and the resulting plasmid is designated as pASMM. Then a human placenta cDNA library was screened using the pASMM as a target plasmid. Seven positive clones were isolated from 150 000 independent transformants. Sequence analysis of one of the positive clones demonstrates that a part of coding sequence of vimentin from 611 bp to 3' end and flanking non-translation region was obtained. Therefore, there is an interaction between vimentin and TPO receptor. The results suggest that cytoskeletal protein may play an important role in TPO signal transduction pathway.     

Mapping of a linear autoantigenic epitope within the human thyroid peroxidase using recombinant DNA techniques.

Autoantibodies directed against the thyroid peroxidase (TPO), the thyroid microsomal antigen, are widely used to diagnose human autoimmune thyroid disease. A cloned 3.088 kb cDNA coding for the entire mature human TPO was isolated from a cDNA library derived from a pathological thyroid gland of a Graves' disease patient and used further to generate a so-called TPO epitope cDNA library in order to map linear autoantigenic epitopes involving a recombinant molecular biology approach. The TPO epitope cDNA library consisting of randomly fragmented cDNA sequences inserted in the expression vector pGEX-2T was expressed in Escherichia coli and screened with characterized anti-TPO autoantisera from Hashimoto's disease patients. All the sera were positively tested with a purified thyroid microsomal antigen fraction (TMA/TPO). Only about 1% of examined autoantisera were able to recognize bacterial expressed recombinant TPO representing sequential antigenic determinants. A corresponding autoantigenic epitope with 61 amino acids in length was located at the C-terminus of human TPO.     

Screening of proteins that interact with human thrombopoietin receptor c-Mpl using yeast two-hybrid system

Thrombopoietin (TPO) is the major cytokine involved in platelet production and exerts its effects via the receptor c-Mpl. The yeast two-hybrid system has been used to screen the proteins interacting with c-Mpl. First, the cDNA fragment of c-Mpl intracellular domain was cloned into two-hybrid vector pAS2, and the resulting plasmid is designated as pASMM. Then a human placenta cDNA library was screened using the pASMM as a target plasmid. Seven positive clones were isolated from 150 000 independent transformants. Sequence analysis of one of the positive clones demonstrates that a part of coding sequence of vimentin from 611 bp to 3′ end and flanking non-translation region was obtained. Therefore, there is an interaction between vimentin and TPO receptor. The results suggest that cytoskeletal protein may play an important role in TPO signal transduction pathway.     

Isolation of Expressed Sequences Encoded by the Human Xq Terminal Portion Using Microclone Probes Generated by Laser Microdissection

The genes that cause a variety of neurologic and neuromuscular disorders have been mapped to the distal region of Xq. In an effort to isolate genes from this area, a regional genomic library of the distal 30% of Xq was constructed from a single metaphase spread by means of laser microdissection and single unique primer-polymerase chain reaction. Using pooled probes of 1000 clones from the genomic library, human brain cDNA libraries were screened for expressed sequences encoded by this region. From the 250,000 cDNA clones screened so far, 10 nonoverlapping sequences that mapped back to the target portion were isolated. The complete nucleotide sequences of these cDNA clones have been determined. Analysis of the sequences indicates that none has significant similarity to previously characterized primate genes. One sequence mapping to Xq27.3-qter contained an open reading frame of 281 amino acids and was expressed in every tissue tested. This gene, as well as others isolated in this manner, may prove to be a candidate gene for heritable disorders mapping to this region.     

Complete amino acid sequence of human cartilage link protein (CRTL1) deduced from cDNA clones and chromosomal assignment of the gene

Little is known about the primary amino acid structure of human cartilage link protein (CRTL1). We screened a human genomic library with a cDNA encoding the 3' untranslated region and the adjoining B1 domain of chicken link protein. One clone was isolated and characterized. A 3.5-kb EcoRI-KpnI fragment from this genomic clone that contains the human B1 exon was used to map the gene to chromosome 5q13----q14.1. The same fragment was used to screen a cDNA library prepared from mRNA of Caco-2, a human colon tumor cell line. Two overlapping clones were isolated and shown to encode all of CRTL1. The deduced amino acid sequence is 354 residues long. The amino acid sequence shows a striking degree of identity to the porcine (96%), rat (96%), and chicken (85%) link protein sequences. Furthermore, there is greater than 86% homology between the 3' untranslated region of the genes encoding human and porcine link proteins. These results indicate that there has been strong evolutionary pressure against changes in the coding and 3' untranslated regions of the gene encoding cartilage link protein.     

Isolation and characterization of full-length cDNA clones for human alpha-, beta-, and gamma-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed.

cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (gamma and beta, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (alpha, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some full-length cDNAs were enriched as much as 50- to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes gamma actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes beta actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes alpha actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis of the DNA sequences of the 5' end of the clones demonstrated that although beta- and gamma-actin genes start with a methionine codon (MET-Asp-Asp-Asp and MET-Glu-Glu-Glu, respectively), the alpha-actin gene starts with a methionine codon followed by a cysteine codon (MET-CYS-Asp-Glu-Asp-Glu). Since no known actin proteins start with a cysteine, it is likely that post-translational removal of cysteine in addition to methionine accompanies alpha-actin synthesis but not beta- and gamma-actin synthesis. This observation has interesting implications both for actin function and actin gene regulation and evolution.     

The mink proopiomelanocortin gene: characterization of cDNA and chromosomal localization

A cDNA library from the mink pituitary was screened using as probe a synthetic oligodeoxyribonucleotide, 5'-TTCATGACCTCCGA-3', corresponding to the endorphin region of bovine proopiomelanocortin (POMC) cDNA. As a result, several clones containing inserts complementary to POMC mRNA were identified. The sequence of one of the fragments (585 bp, 65% of the total length of mRNA) was determined. A high degree of homology (over 80%) among the primary structures of sequences from mink, man, and bovine cDNA POMC was established. With the cloned mink cDNA fragment as probe, the DNAs from mink-Chinese hamster hybrid clones were studied. The results of segregation analysis of mink POMC sequences and mink chromosomes in the mink-Chinese hamster panel allowed us to assign the POMC gene to mink chromosome 11.     

Molecular cloning and characterization of a full-length cDNA clone for human plasminogen

A human liver cDNA library enriched for full-length clones was screened for plasminogen cDNA using a synthetic 24-nucleotide probe derived from a reported partial cDNA sequence. 12 positive clones were identified and one of these was characterized in detail. The 2.7 kb insert contains the complete coding region. At 5 positions, it gives residues different from those reported in a previous amino acid sequence analysis of the protein. The present results show an extra Ile at position 65, Gln instead of Glu at positions 53 and 342, Asn at position 88 instead of Asp, and Asp at position 453 rather than Asn. In the 3'-non-coding region an extension of 29 bases is found which does not contain any structure compatible with a known polyadenylation signal. Instead, the consensus signal AATAAA is placed at a distance of 46 bases upstream of the poly(A)-tail.     

Cloning and expression of murine lymphotoxin cDNA

The murine lymphotoxin (LT) gene has been cloned and used to identify cDNA clones in a library prepared from activated murine T cell mRNA. A recombinant murine genomic library was screened with a human lymphotoxin cDNA probe, resulting in the isolation of the entire LT gene. The murine LT gene structure is similar to the human gene, containing three intervening sequences. An activated murine T cell cDNA library was prepared with poly(A)+ RNA isolated 7 hr after concanavalin A stimulation of an L3T4+ interleukin 2-dependent murine T cell clone. Two colonies of the cDNA library that contained inserts that hybridized with the murine LT gene probe were sequenced and were used to construct expression plasmids. The amino acid sequence deduced from the cDNA indicates that murine LT is highly homologous to human LT (74%) and is related to murine tumor necrosis factor (35% homology). The cDNA was transcribed and was translated in vitro, and was expressed in COS-1 cells. This has resulted in the production of LT biological activity.     

Isolation and characterization of a cDNA clone for porcine thyroid peroxidase

We undertook the molecular cloning of porcine thyroid peroxidase (TPO). Four oligonucleotide probes were synthesized on the basis of amino acid sequences of 3 tryptic peptides from highly purified porcine TPO. These probes were used to screen a pig thyroid cDNA library. Seven of 16 selected clones (0.45-1.15 kb in size) reacted with all 4 probes. Nucleotide sequencing of the 1.15 kb at the 3'-end of the structural gene revealed the complementary sequence to all 4 probes as well as the nucleotides coding for the entire length of the 3 tryptic peptides. There is an open reading frame of 332 amino acid residues. On Northern blot analysis this gene codes for an mRNA species of 2.85 kb, corresponding to the anticipated size of the mRNA for the intact TPO molecule. We have therefore cloned and characterized a cDNA clone coding for approx. 36% of porcine thyroid peroxidase.     

Analysis of cDNA and genomic clones coding for the pro alpha 1 chain of calf type II collagen.

A bovine cDNA library constructed from fetal cartilage RNA was screened with a pro alpha 1(II) collagen specific chicken cDNA. A recombinant clone (Bc 7), with an insert of 1 kb, was identified and shown to contain sequences exhibiting 85% homology with the chicken pro alpha 1(II) collagen C-propeptide. Interspecies comparison strongly suggested that one potential glycosylation site present in the avian C-propeptide is not utilized, since this site is absent in the bovine chain. In addition, two overlapping genomic clones (Pal 3 and Pal 4) were isolated and partially characterized. These clones span 23 kb of DNA and contain approximately 17 kb of the pro alpha 1(II) calf gene. Sequencing of exon 1 has determined the length of the 3' untranslated region and the exact location of the polyadenylation attachment site.