Human Muscle cDNA Library Research Articles

Expression of the functional carbohydrate-binding module (CBM) of human laforin

Laforin is a human protein associated with the glycogen metabolism, composed of two structurally and functionally independent domains: a phosphatase catalytic domain and a substrate-binding module with glycogen and starch affinity. The main goal of this work is the development of a methodology for the expression of the so far poorly characterized carbohydrate-binding module (CBM) of laforin, allowing its study and development of biomedical applications. The laforin’s CBM sequence was originally cloned by PCR from a human muscle cDNA library. The recombinant protein, containing laforin’s CBM fused to an Arg-Gly-Asp sequence (RGD), was cloned and expressed using vector pET29a and recovered as inclusion bodies (IBs). Refolding of the IBs allowed the purification of soluble, dimeric and functional protein, according to adsorption assays using starch and glycogen. Several other experimental approaches, using both bacteria and yeast, were unsuccessfully tested, pointing towards the difficulties in producing the heterologous protein. Indeed, this is the first work reporting the production of the functional CBM from human laforin.

Cloning and Characterization of KCC3 and KCC4, New Members of the Cation-Chloride Cotransporter Gene Family

The K+-Cl- cotransporters (KCCs) belong to the gene family of electroneutral cation-chloride cotransporters, which also includes two bumetanide-sensitive Na+-K+-2Cl- cotransporters and a thiazide-sensitive Na+-Cl- cotransporter. We have cloned cDNAs encoding mouse KCC3, human KCC3, and human KCC4, three new members of this gene family. The KCC3 and KCC4 cDNAs predict proteins of 1083 and 1150 amino acids, respectively. The KCC3 and KCC4 proteins are 65-71% identical to the previously characterized transporters KCC1 and KCC2, with which they share a predicted membrane topology. The four KCC proteins differ at amino acid residues within key transmembrane domains and in the distribution of putative phosphorylation sites within the amino- and carboxyl-terminal cytoplasmic domains. The expression of mouse KCC3 in Xenopus laevis oocytes reveals the expected functional characteristics of a K+Cl- cotransporter: Cl--dependent uptake of 86Rb+ which is strongly activated by cell swelling and weakly sensitive to furosemide. A direct functional comparison of mouse KCC3 to rabbit KCC1 indicates that KCC3 has a much greater volume sensitivity. The human KCC3 and KCC4 genes are located on chromosomes 5p15 and 15q14, respectively. Although widely expressed, KCC3 transcripts are the most abundant in heart and kidney, and KCC4 is expressed in muscle, brain, lung, heart, and kidney. The unexpected molecular heterogeneity of K+-Cl- cotransport has implications for the physiology and pathophysiology of a number of tissues.

The molecular basis for cross-reaction of an anti-dystrophin antibody with alpha-actinin

The epitope recognised by the anti-dystrophin monoclonal antibodies MANDYS141 and MANDYS142 has been characterised using a phage display peptide library and a bacteriophage lambda cDNA library. Using a phage display library of random 15-mer peptides, the epitope recognised by the two antibodies was identified as EEXF. A λgt11 clone obtained by screening a human muscle cDNA library was shown to contain part of the out-of-frame human mitochondrial succinyl CoA synthetase (α-subunit) cDNA sequence which contains the sequence EEPL, suggesting a minimum requirement of EEXF/L for antibody binding. The sequence EEDF is located in the helical rod region of dystrophin and the N-terminal domain of α-actinin; this may explain why native dystrophin is not detected, since the α-helical, coiled-coil folding of the rod region of dystrophin may obscure the epitope in the native protein. The antibody cross-reaction between dystrophin and α-actinin is likely to be fortuitous and not due to any structural homology that exists between these two members of the spectrin superfamily.

Yeast two-hybrid cloning of a novel zinc finger protein that interacts with the multifunctional transcription factor YY1.

Muscle-restricted transcription of sarcomeric actin genes is negatively controlled by the zinc finger protein YY1, which is down-regulated at the protein level during myogenic differentiation. To identify cellular proteins that might mediate the function/stability of YY1 in muscle cells, we screened an adult human muscle cDNA library using the yeast two-hybrid cloning system. We report the isolation and characterization of a novel protein termed YAF2 (YY1- associated factor 2) that interacts with YY1. The YAF2 cDNA encodes a 180 amino acid basic protein (pI 10.5) containing a single N-terminal C2-X10-C2 zinc finger. Lysine clusters are present that may function as a nuclear localization signal. Domain mapping analysis shows that the first and second zinc fingers of YY1 are targeted for YAF2 protein interaction. In contrast to the down-regulation of YY1, YAF2 message levels increase during in vitro differentiation of both rat skeletal and cardiac muscle cells. YAF2 appears to have a promyogenic regulatory role, since overexpression of YAF2 in C2 myoblasts stimulates myogenic promoter activity normally restricted by YY1. Co-transfection of YY1 reverses the stimulatory effect of YAF2. YAF2 also greatly potentiates proteolytic cleavage of YY1 by the calcium- activated protease m-calpain. The isolation of YAF2 may help in understanding the mechanisms through which inhibitors of myogenic transcription may be antagonized or eliminated by proteolysis during muscle development.

A New Human Slow Skeletal Troponin T (TnTs) mRNA Isoform Derived from Alternative Splicing of a Single Gene

A human muscle cDNA library was screened for slow skeletal troponin T (TnTs). Sequence analysis revealed that one of the selected clones had a cDNA coding sequence different from the two previously described. RNase protection assays confirmed the expression of this new isoform in adult skeletal muscle. In addition, results of the RNase protection assays strongly suggested the expression of a fourth isoform. These isoforms for human slow TnT most likely result from combinatorial alternative splicing of a single gene.

93 MOLECULAR ASPECT OF MYOGENIC HYPERURICEMIA: CLONING OF HUMAN MUSCLE PHOSPHOFRUCTOKINASE cDNA

Our recent investigations revealed that myogenic hyperuricemia (Mineo et al.) is a common pathologic feature of glycogenosis types III, V and VII, in which muscle glycolysis is impaired. To clarify the molecular defect in glycogenosis type VII (human muscle phosphofructokinase (HMPFK) deficiency), we began performing the molecular cloning of HMPFK cDNA. A human muscle cDNA library was prepared by Okayama and Berg method and screened by colony hybridization with a rabbit muscle PFK cDNA (a gift from Dr. S. D. Putney of the Repligen Corp. MA, U.S.A.). A partial HMPFK cDNA clone was isolated. The clone was primer-extended and two other overlapping clones to cover the full-length were isolated. Complete primary structure of the enzyme was determined through the sequence analysis. HMPFK was 85,050 Da in size with 779 amino acid residues. Internal homology between N- and C- halves of the peptide was observed as in rabbit muscle PFK. Sequence homologies between rabbit and human muscle PFK were 96% in amino acids and 89% in nucleotides. Cloning of HMPFK cDNA will help analyzing the pathological regulatory mechanism of muscle glycolysis responsible for the development of myogenic hyperuricemia at the molecular level.

Fast-twitch and slow-twitch/cardiac Ca2+ ATPase genes map to human chromosomes 16 and 12.

The fast-twitch and slow-twitch/cardiac Ca2+ ATPase genes have been assigned to human chromosomes 16 and 12, respectively, using rodent-human somatic cell hybrids and filter hybridization analysis of cell hybrid DNA. A rabbit cDNA for the fast-twitch ATPase hybridizes to a prominent single fragment in human genomic DNA digested with the restriction enzyme BamHI. By correlating the presence of this fragment in somatic cell hybrid DNA with the human chromosome content of the hybrids, the fast-twitch ATPase gene can be assigned to human chromosome 16. A slow-twitch/cardiac ATPase cDNA clone was isolated from a human muscle cDNA library and used to detect human fragments in EcoRI-digested somatic cell hybrid DNA. By correlating the presence of these fragments with the human chromosome content of the hybrids, the slow-twitch/cardiac ATPase gene can be assigned to human chromosome 12. Thus, the two ATPase genes, which are probably related to each other by an ancient duplication event, are not syntenic in the human genome.

Nucleotide sequence, tissue-specific expression, and chromosome location of human carbonic anhydrase III: the human CAIII gene is located on the same chromosome as the closely linked CAI and CAII genes.

The carbonic anhydrases (CA) are a class of metalloenzymes that catalyze the reversible hydration of carbon dioxide. The genes for the carbonic anhydrase isozymes are members of a multigene family that are differentially expressed in a number of cell types. We have isolated a full-length representative of a CAIII mRNA transcript from an adult human muscle cDNA library, and we present the complete nucleotide sequence of this cDNA clone. RNA blots demonstrate that CAIII messages can be detected in a variety of cell types but that high-level expression is limited to human fetal and adult skeletal muscle and to rodent slow skeletal muscle and liver. In addition, we have used a panel of human-mouse cell hybrids to localize the human CAIII gene to chromosome 8. Previous reports have established the CAI and CAII isozyme genes to be closely linked on chromosome 8, and the assignment of the CAIII gene to the same chromosome raises the possibility that these genes may all be linked at a single complex locus.

Isolation of repetitive clones from human muscle cDNA library.

Human fetal muscle cDNA library was screened with a beta-myosin heavy chain gene fragment containing Alu sequences. Two cDNA clones AI and BII with 1.8 and 3 kb inserts respectively were chosen for further characterization by means of RNA and DNA hybridization procedures and sequencing. The clones appeared to contain repetitive sequences as well as single copy regions. They are actively transcribed in different stages of myogenic development but not in the liver. DNA sequence analysis of short stretches from both clones revealed no sequence homology to any other published DNA sequences.

Isolation of a cDNA clone for the human muscle specific carbonic anhydrase, CAIII.

The molecular cloning of cDNA for the human muscle specific carbonic anhydrase CAIII is described. The recombinant was isolated from a human muscle cDNA library prepared in the expression vector lambda gt11, and was characterized by hybridization selection and immunoprecipitation. A comparison of insert cDNA and mRNA sizes suggests that the cDNA is full length and includes extensive untranslated sequences. Preliminary sequence data have confirmed the authenticity of this clone and Southern blotting of human and rodent DNA indicates that it will be a useful probe in the analysis of somatic cell hybrids.

Alpha-skeletal and alpha-cardiac actin genes are coexpressed in adult human skeletal muscle and heart.

We determined the actin isotypes encoded by 30 actin cDNA clones previously isolated from an adult human muscle cDNA library. Using 3' untranslated region probes derived from alpha-skeletal, beta- and gamma-actin cDNAs and from an alpha-cardiac actin genomic clone, we showed that 28 of the cDNAs correspond to alpha-skeletal actin transcripts. Unexpectedly, however, the remaining two cDNA clones proved to derive from alpha-cardiac actin mRNA. Sequence analysis confirmed that the two skeletal muscle alpha-cardiac actin cDNAs are derived from transcripts of the cloned alpha-cardiac actin gene. Direct measurements of actin isotype mRNA expression in human skeletal muscle showed that alpha-cardiac actin mRNA is expressed at 5% the level of alpha-skeletal actin. Furthermore, the alpha-cardiac actin gene expressed in skeletal muscle is the same gene which produces alpha-cardiac actin mRNA in the human heart. Of equal surprise, we found that alpha-skeletal actin mRNA accounts for about half of the total actin mRNA in adult heart. Comparison of total actin mRNA levels in adult skeletal muscle and adult heart revealed that the steady-state levels in skeletal muscle are about twofold greater, per microgram of total cellular RNA, than those in heart. Thus, in skeletal muscle and in heart, both of the sarcomeric actin mRNA isotypes are quite abundant transcripts. We conclude that alpha-skeletal and alpha-cardiac actin genes are coexpressed as an actin pair in human adult striated muscles. Since the smooth-muscle actins (aortic and stomach) and the cytoplasmic actins (beta and gamma) are known to be coexpressed in smooth muscle and nonmuscle cells, respectively, we postulate that coexpression of actin pairs may be a common feature of mammalian actin gene expression in all tissues.

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.

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.