The Characterization of Alpha- and Beta-Carbonic Anhydrases of Arabidopsis thaliana

Carbonic anhydrases (CAs) are zinc-metalloenzymes that interconvert two inorganic carbon (Ci) species, CO2 and HCO3-. In Arabidopsis thaliana, there are eight Alpha-CA genes, six Beta-CA genes, three Gamma-CA genes, and two Gamma-CA-like genes. The majority of CA research in plants has focused on finding a link between CA activity and photosynthesis rates. Since the CA genes are expressed in different plant tissues and multiple CA isoforms are distributed among various organelles of the plant cell, I hypothesize that CAs facilitate CO2 diffusion among cell compartments and maintain Ci pools for carbon-requiring reactions by interconverting CO2 and HCO3-. This thesis focuses on the Alpha-CAs and Beta-CAs of Arabidopsis and how they may affect various reactions throughout the plant. CA T-DNA insertion lines were used to determine if removing one or more CAs from Arabidopsis affects the plant growth. The Beta-ca5 single mutant and Beta-ca2Beta-ca4, Alpha-ca1Beta-ca4, and Alpha-ca2Beta-ca4 double mutants show different growth phenotypes. The Beta-ca2Beta-ca4 plants were smaller in size and chlorotic in their younger leaves under low CO2 conditions, but showed improved growth in high CO2 conditions. The growth of the Beta-ca5 single mutant was severely stunted in ambient CO2 conditions and high CO2 partially rescued wildtype growth in the Beta-ca5 plants. The Alpha-ca1Beta-ca4 and Alpha-ca2Beta-ca4 double mutants were slightly smaller than wildtype plants in low CO2 conditions. Interestingly, it seems the reduced growth of the Beta-ca5 single mutant and Beta-ca2Beta-ca4 double mutant plants was not linked to deficiencies in photosynthesis rates but rather may be required for other carbon requiring reactions. These results suggest that CAs are playing more complex roles in plants than once thought and that the various isoforms are affecting different carbon-requiring pathways

Both Myoblast Lineage and Innervation Determine Fiber Type and Are Required for Expression of the Slow Myosin Heavy Chain 2 Gene

AbstractSkeletal muscle fibers express members of the myosin heavy chain (MyHC) gene family in a fiber-type-specific manner. In avian skeletal muscle it is the expression of the slow MyHC isoforms that most clearly distinguishes slow- from fast-contracting fiber types. Two hypotheses have been proposed to explain fiber-type-specific expression of distinct MyHC genes during development—an intrinsic mechanism based on the formation of different myogenic lineage(s) and an extrinsic, innervation-dependent mechanism. We developed a cell culture model system in which both mechanisms were evaluated during fetal muscle development. Myoblasts isolated from prospective fast (pectoralis major) or slow (medial adductor) fetal chick muscles formed muscle fibers in cell culture, none of which expressed slow MyHC genes. By contrast, when muscle fibers formed from myoblasts derived from the slow muscle were cocultured with neural tube, the muscle fibers expressed a slow MyHC gene, while muscle fibers formed from myoblasts of fast muscle origin continued to express only fast MyHC. Motor endplates formed on the fibers derived from myoblasts of both fast and slow muscle origin in cocultures, and slow MyHC gene expression did not occur when neuromuscular transmission or depolarization was blocked. We have cloned the slow MyHC gene that is expressed in response to innervation and identified it as the slow MyHC 2 gene, the predominant adult slow isoform. cDNAs encoding portions of the three slow myosin heavy chain genes (MyHC1, slow MyHC 2, and slow MyHC 3) were isolated. Only slow MyHC 2 mRNA was demonstrated to be abundant in the cocultures of neural tube and muscle fibers derived from myoblasts of slow muscle origin. Thus, expression of the slow MyHC 2 gene in thisin vitrosystem indicates that formation of slow muscle fiber types is dependent on both myoblast lineage (intrinsic mechanisms) and innervation (extrinsic mechanisms), and suggests neither mechanism alone is sufficient to explain formation of muscle fibers of different types during fetal development

The Nopp140 gene in Drosophila melanogaster displays length polymorphisms in its large repetitive second exon

Nopp140, often called the nucleolar and Cajal body phosphoprotein (NOLC1), is an evolutionarily conserved chaperone for the transcription and processing of rRNA during ribosome subunit assembly. Metazoan Nopp140 contains an amino terminal LisH dimerization domain and a highly conserved carboxyl domain. A large central domain consists of alternating basic and acidic motifs of low sequence complexity. Orthologous versions of Nopp140 contain variable numbers of repeating basic-acidic units. While vertebrate Nopp140 genes use multiple exons to encode the central domain, the Nopp140 gene in Drosophila uses exclusively exon 2 to encode the central domain. Here, we define three overlapping repeat sequence patterns (P, P\u27, and P \u27\u27) within the central domain of D. melanogaster Nopp140. These repeat patterns are poorly conserved in other Drosophila species. We also describe a length polymorphism in exon 2 that pertains specifically to the P\u27 pattern in D. melanogaster. The pattern displays either two or three 96 base pair repeats, respectively, referred to as Nopp140-Short and Nopp140-Long. Fly lines homozygous for one or the other allele, or heterozygous for both alleles, show no discernible phenotypes. PCR characterization of the long and short alleles shows a poorly defined, artifactual bias toward amplifying the long allele over the short allele. The significance of this polymorphism will be in discerning the largely unknown properties of Nopp140\u27s large central domain in rDNA transcription and ribosome biogenesis