Dipak K. Dube

Ectopic expression of tropomyosin promotes myofibrillogenesis in mutant axolotl hearts

Expression of tropomyosin protein, an essential component of the thin filament, has been found to be drastically reduced in cardiac mutant hearts of the Mexican axolotl (Ambystoma mexicanum) with no formation of sarcomeric myofibrils. Therefore, this naturally occurring cardiac mutation is an appropriate model to examine the effects of delivering tropomyosin protein or tropomyosin cDNA into the deficient tissue. In this study, we describe the replacement of tropomyosin by using a cationic liposome transfection technique applied to whole hearts in vitro. When mouse α‐tropomyosin cDNA under the control of a cardiac‐specific α‐myosin heavy chain promoter was transfected into the mutant hearts, tropomyosin expression was enhanced resulting in the formation of well‐organized sarcomeric myofibrils. Transfection of a β‐tropomyosin construct under control of the same promoter did not result in enhanced organization of the myofibrils. Transfection of a β‐galactosidase reporter gene did not result in the formation of organized myofibrils or increased tropomyosin expression. These results demonstrate the importance of α‐tropomyosin to the phenotype of this mutation and to normal myofibril formation. Moreover, we have shown that a crucial contractile protein can be ectopically expressed in cardiac muscle that is deficient in this protein, with the resulting formation of organized sarcomeres. Dev. Dyn. 1998;213:412–420.

Differential expression of a novel isoform of α-tropomyosin in cardiac and skeletal muscle of the Mexican axolotl (Ambystoma mexicanum)

Alternative mRNA splicing is a fundamental process in eukaryotes that contributes to tissue-specific and developmentally regulated patterns of tropomyosin (TM) gene expression. Northern blot analyses suggest the presence of multiple transcripts of tropomyosin in skeletal and cardiac muscle of adult Mexican axolotls. We have cloned and sequenced two tropomyosin cDNAs designated ATmC-1 and ATmC-2 from axolotl heart tissue and one TM cDNA from skeletal muscle, designated ATmS-1. Nucleotide sequence analyses suggest that ATmC-1 and ATmC-2 are the products of the same alpha-TM gene produced via alternate splicing, whereas ATmC-1 and ATmS-1 are the identical isoforms generated from the alpha-gene. RT-PCR analysis using isoform-specific primer pairs and detector oligonucleotides suggests that ATmC-2 is expressed predominantly in adult axolotl hearts. ATmC-2 is a novel isoform, which unlike ATmC-1 and other known striated muscle isoforms expresses exon 2a instead of exon 2b.

Selection of new biologically active molecules from random nucleotide sequences

Genetic diversity can be achieved in vitro by inserting random nucleotide (nt) sequences into cloned genes. In the case of enzymes, subsequent genetic complementation can be used to select for new mutants that exhibit different substrate specificities, altered catalytic activities, or altered temperature sensitivities. Using this technique, one can also analyze the contribution of different amino acid residues to the structure and function of enzyme. Selecting biologically active DNA sequences from large random populations provides a new method for identifying nt sequences with unique functions. Analogous random sequence selection techniques have been applied to determine the consensus sequence of the Escherichia coli promoters, DNA and RNA sequences that bind specific protein(s), DNA regulatory sequences, ribozyme(s) and ligand-specific RNA(s). In this manuscript, we will consider recent data obtained in our laboratory as a result of inserting random sequences into the putative nucleoside-binding site of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK). We have obtained over 2000 new mutant HSV-1 TKs, some of which are stable at higher temperatures or have altered substrate specificity and/or catalytic rates when compared to those of the wild-type enzyme.

Identification, characterization, and expression of a novel α‐tropomyosin isoform in cardiac tissues in developing chicken

Tropomyosins are present in various muscle (skeletal, cardiac, and smooth) and non‐muscle cells with different isoforms characteristic of specific cell types. We describe here a novel smooth/striated chimeric isoform that was expressed in developing chick heart in addition to the classically described TM‐4 type. This novel α‐Tm tropomyosin isoform, designated as α‐Tm‐2, contains exon 2a (in place of exon 2b). The known striated muscle isoform (α‐Tm‐1) was also expressed in embryonic hearts along with the striated muscle isoform of TM‐4. In adult heart, TM‐4 was expressed, however, expression of both α‐Tm‐1 and α‐Tm‐2 isoforms was drastically reduced or downregulated. Interestingly, we were unable to detect the expression of α‐Tm‐2 in embryonic and adult skeletal muscle, however, the α‐Tm‐1 isoform is expressed in embryonic and adult skeletal muscle. Examination of other possible isoforms of the α‐TM gene, i.e., α‐smooth muscle tropomyosin (α‐Sm), α‐Fibroblast‐1 (α‐F1), and α‐Fibroblast‐2 (α‐F2) revealed expression in embryonic hearts and a significant reduction of each of these isoforms in adult heart. In order to elucidate the role of the newly discovered tropomyosin isoform in chicken, we ectopically expressed the GFP fusion protein of α‐Tm‐1 and α‐Tm‐2 separately into cardiomyocytes isolated from neonatal rats. Each isoform was incorporated into organized myofibrils. Our results suggest that the α‐TM gene may undergo both positive and negative transcriptional control in chicken hearts during development.

Alteration of cardiac myofibrillogenesis by liposome-mediated delivery of exogenous proteins and nucleic acids into whole embryonic hearts

A precise organization of contractile proteins is essential for contraction of heart muscle. Without a necessary stoichiometry of proteins, beating is not possible. Disruption of this organization can be seen in diseases such as familial hypertrophic cardiomyopathy and also in acquired diseases. In addition, isoform diversity may affect contractile properties in such functional adaptations as cardiac hypertrophy. The Mexican axolotl provides an uncommon model in which to examine specific proteins involved with myofibril formation in the heart. Cardiac mutant embryos lack organized myofibrils and have altered expression of contractile proteins. In order to replicate the disruption of myofibril formation seen in mutant hearts, we have developed procedures for the introduction of contractile protein antibodies into normal hearts. Oligonucleotides specific to axolotl tropomyosin isoforms (ATmC-1 and ATmC-3), were also successfully introduced into the normal hearts. The antisense ATmC-3 oligonucleotide disrupted myofibril formation and beating, while the sense strands did not. A fluorescein-tagged sense oligonucleotide clearly showed that the oligonucleotide is introduced within the cells of the intact hearts. In contrast, ATmC-1 anti-sense oligonucleotide did not cause a disruption of the myofibrillar organization. Specifically, tropomyosin expression can be disrupted in normal hearts with a lack of organized myofibrils. In a broader approach, these procedures for whole hearts are important for studying myofibril formation in normal hearts at the DNA, RNA, and/or protein levels and can complement the studies of the cardiac mutant phenotype. All of these tools taken together present a powerful approach to the elucidation of myofibrillogenesis and show that embryonic heart cells can incorporate a wide variety of molecules with cationic liposomes.

Expression of axolotl RNA-binding protein during development of the Mexican axolotl

Abstract Amphibians occupy a central position in phylogeny between aquatic and terrestrial vertebrates and are widely used as model systems for studying vertebrate development. We have undertaken a comprehensive molecular approach to understand the early events related to embryonic development in the Mexican axolotl, Ambystoma mexicanum, which is an exquisite animal model for such explorations. Axolotl RBP is a RNA-binding protein which was isolated from the embryonic Mexican axolotl by subtraction hybridization and was found to show highest similarity with human, mouse, and Xenopus cold-inducible RNA-binding protein (CIRP). The reverse transcriptase polymerase chain reaction (RT-PCR) analysis suggests that it is expressed in most of the axolotl tissues except liver; the expression level appears to be highest in adult brain. We have also determined the temporal and spatial pattern of its expression at various stages of development. RT-PCR and in situ hybridization analyses indicate that expression of the AxRBP gene starts at stage 10–12 (gastrula), reaches a maxima around stage 15–20 (early tailbud), and then gradually declines through stage 40 (hatching). In situ hybridization suggests that the expression is at a maximum in neural plate and neural fold at stage 15 (neurula) of embryonic development.

Differential expression of C-protein isoforms in the developing heart of normal and cardiac lethal mutant axolotls (Ambystoma mexicanum).

Regulated assembly of contractile proteins into sarcomeric structures, such as A‐ and I‐bands, is still currently being defined. The presence of distinct isoforms of several muscle proteins suggests a possible mechanism by which myocytes regulate assembly during myofibrillo‐genesis. Of several muscle isoforms located within the A‐band, myosin binding proteins (MyBP) are reported to be involved in the regulation and stabilization of thick filaments during sarcomere assembly. The present confocal study characterizes the expression of one of these myosin binding proteins, C‐protein (MyBP‐C) in wild‐type and cardiac lethal mutant embryos of the axolotl, Ambystoma mexicanum. C‐protein isoforms are also detected in distinct temporal patterns in whole‐mounted heart tubes and thoracic skeletal muscles. Confocal analysis of axolotl embryos shows both cardiac and skeletal muscles to regulate the expression of C‐protein isoforms over a specific developmental window. Although the CPROAxslow isoform is present during the initial heartbeat stage, its expression is not retained in the adult heart. C‐protein isoforms are simultaneously expressed in both cardiac and skeletal muscle during embryogenesis.

Identification and expression of a homologue of the murine HoxA5 gene in the Mexican axolotl (ambystoma mexicanum)

An excellent model for studying heart development in vertebrates is the cardiac non-function lethal mutant (gene c) Mexican axolotl, Ambystoma mexicanum. In order to facilitate our analyses of the mutant system, we have undertaken a search for stage-specific molecular markers during embryonic development of the axolotl. We have concentrated on homeobox genes as suitable candidates for monitoring molecular changes during development. A 270-bp probe encoding a portion of the axolotl homeobox gene Ahox-1 was generated by PCR from a stage-18 axolotl embryonic cDNA library. 32P-labelled PCR-amplified Ahox-1 DNA was used as the probe for screening a lambda AM18 cDNA library using moderately stringent conditions. We isolated six clones and determined their partial nucleotide (nt) sequences. One of the clones, which has very high homology to human, mouse and rat Hox A5 (83 and 99% at the nt and amino-acid levels, respectively, in the homeodomain region), was analyzed further. RT-PCR analyses show that the level of expression of HoxA5 is very low at stage 11 of embryonic development (gastrula). The level of expression reaches maximum at stage 25 (tailbud) and then plateaus at stages 30 and 35 (heartbeat onset). Although the expression of Ahox-1 was also found to start at stage 11, it reaches a maximum level at stage 25 and declines at stage 35. We have also studied, using RT-PCR, the tissue-specific expression of HoxA5 and Ahox-1 in juvenile axolotl.

Immunohistochemical analysis of C-protein isoforms in cardiac and skeletal muscle of the axolotl, Ambystoma mexicanum.

Of the several proteins located within sarcomeric A-bands, C-protein, a myosin binding protein (MyBP) is thought to regulate and stabilize thick filaments during assembly. This paper reports the characterization of C-protein isoforms in juvenile and adult axolotls, Ambystoma mexicanum, by means of immunofluorescent microscopy and Western blot analyses. C-protein and myosin are found specifically within the A-bands, whereas tropomyosin and α-actin are detected in the I-bands of axolotl myofibrils. The MF1 antibody prepared against the fast skeletal muscle isoform of chicken C-protein specifically recognizes a cardiac isoform (Axcard1) in juvenile and adult axolotls but does not label axolotl skeletal muscle. The ALD66 antibody, which reacts with the C-protein slow isoform in chicken, localizes only in skeletal muscle of the axolotl. This slow axolotl isoform (Axslow) displays a heterogeneous distribution in fibers of dorsalis trunci skeletal muscle. The C315 antibody against the chicken C-protein cardiac isoform identifies a second axolotl cardiac isoform (Axcard2), which is present also in axolotl skeletal muscle. No C-protein was detected in smooth muscle of the juvenile and adult axolotl with these antibodies.

[25] Use of PCR in detection of antisense transcripts in HTLV-I-infected patients and human T-cell lines

Publisher Summary This chapter describes a sensitive and specific polymerase chain reaction (PCR)-based method to detect both (+) and (–) strand human T-cell lymphoma/leukemia viruses (HTLV-I) transcript in DNase-treated, poly(A) RNA from HTLV-I-infected cell cultures via reverse transcriptase-directed polymerase chain reaction (RT-PCR), using the thermostable enzyme from Thermus thermophilus , rTth, and various primer oligonucleotides in sequential fashion. Classically, human retroviruses, including HTLV-I, contain diploid copies of positive [(+)] or sense strand RNA as their genome. On the infection of the target cell, this RNA is reverse-transcribed into double-stranded proviral DNA that integrates into the host chromosomal DNA and/or exists as unintegrated viral DNA serving as a template for subsequent viral RNA transcription. The issue of negative strand or antisense viral or host cellular RNA synthesis in retrovirus-infected cells has been raised by several investigators. The strategy for detecting (+) or (–) strand RNA is shown in the chapter. Poly(A) RNA is first reverse-transcribed into complementary DNA and then amplified using the DNA polymerase, rTth .