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Studies of Gene Structure: I. Expression of Human α-Globin Genes in COS Cells. II. Isolation and Characterization of the Myosin Light Chain Genes from Drosophila melanogaster

Citation

Parker, Vann Phillips (1985) Studies of Gene Structure: I. Expression of Human α-Globin Genes in COS Cells. II. Isolation and Characterization of the Myosin Light Chain Genes from Drosophila melanogaster. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/msv6-0s82. https://resolver.caltech.edu/CaltechTHESIS:01222019-155306456

Abstract

The first part of this thesis describes the establishment of COS cells as an effective host for the replication and expression of genes cloned into plasmids containing the SV40 viral replication origin. The human α-globin gene was cloned into a pBR322 derivative which contained a 311 base pair SV40 fragment. These plasmids were replicated to high copy number. The α-globin gene was transcribed to produce high levels of RNA which was indistinguishable from human α-globin mRNA. Deletion mutants were generated which defined the region from 55 to 87 base pairs upstream from the mRNA cap site as necessary for high levels of transcription of this gene. This region includes the conserved sequence CCAAT found in a similar position upstream from many eukaryotic genes.

The second part of this thesis describes the cloning of muscle specific genes from Drosophila melanogaster and a detailed analysis of the two myosin light chain genes. A genomic library was screened with RNA isolated from the late pupal stage of development. Recombinant DNA clones which mapped to 24 independent locations were identified. The clones containing the myosin light chain genes were identified from within this group. A cDNA library was generated from late pupal RNA. Clones homologous to each of the myosin light chains were identified and their cloned inserts were sequenced. Their classification as Drosophila myosin light chain genes was established by comparing the derived amino acid sequence to the protein sequence of the chicken myosin light chains. Each myosin light chain is single copy. The alkali myosin light chain maps to the chromosomal locus 98B. It hybrid selects RNA which may be translated in vitro to yeild several polypeptides of molecular weight 18,000 to 19,000 daltons. The gene for myosin light chain-2 maps to the chromosomal locus 99E and is shown to encode two proteins of apparent molecular weights 26,000 and 17,000 daltons. Each protein has a putative divalent cation binding domain.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Biology
Degree Grantor:California Institute of Technology
Division:Biology
Major Option:Biology
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Davidson, Norman R.
Thesis Committee:
  • Attardi, Giuseppe (chair)
  • Hood, Leroy E.
  • Meyerowitz, Elliot M.
  • Strauss, James H.
  • Davidson, Norman R.
Defense Date:9 January 1985
Funders:
Funding AgencyGrant Number
NSFUNSPECIFIED
NIHUNSPECIFIED
Record Number:CaltechTHESIS:01222019-155306456
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:01222019-155306456
DOI:10.7907/msv6-0s82
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/0092-8674(81)90411-6DOIArticle adapted for Ch.1
https://doi.org/10.1128/mcb.4.5.956DOIArticle adapted for Ch. 2
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:11356
Collection:CaltechTHESIS
Deposited By: Melissa Ray
Deposited On:29 Jan 2019 19:06
Last Modified:19 Apr 2021 22:29

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