Date of Award:

5-1-1993

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Biology

Department name when degree awarded

Biology

Committee Chair(s)

Gregory J. Podgorski

Committee

Gregory J. Podgorski

Committee

Reed Warren

Committee

Dennis Welker

Abstract

This thesis reports the molecular cloning, sequence analysis, and developmental expression of a novel Dictyostelium discoideum gene, SQ1. The SQ1 gene was isolated as a set of overlapping cDNA and genomic clones. The longest of these is a 1.5 kb genomic DNA that contains the entire 3, portion of the coding region, and a cDNA that extends the available sequence 0.7 kb 5' of the genomic DNA. The SQ1 mRNA is 2.7 kb and the available cloned sequence is 2239 nucleotides. By assuming a 3, untranslated region of 100 nucleotides, approximately 400 nucleotides of transcribed sequence remain uncloned. The complete 5' region could not be isolated despite repeated attempts using a variety of methods. This may reflect some unusual structure in this region that prevents its efficient replication in cDNA or PCR synthesis or that prohibits its maintenance in E. coli. The SQ1 gene is transcribed to produce a single 2.7 kb mRNA during multicellular stages of development. It first appears by 12 h, when multicellular aggregates were in the process of tip formation, and was still detectable by 16 h, the beginning of culmination. After this stage of development, SQ1 mRNA disappeared rapidly. In shaken suspension SQ1 mRNA does not appear unless cAMP is added to the cells. Under these conditions, the SQ1 mRNA appears much earlier, by 2 h, than in cells developing on filters. In contrast to many Dictyostelium developmentally regulated genes that are only induced by pulses of cAMP and repressed by a constant high amount, SQ1 message is induced by both high levels and pulses of cAMP. The SQ1 mRNA is nonabundant under any conditions of development or cAMP stimulation. The partial SQ1 gene clone contains a single open reading frame that encodes a protein of 720 amino acids. Codon usage corresponds to other Dictyostelium genes in which there is a strong bias for A or T residues. The coding sequence is terminated with a TAA stop codon, as observed for the great majority of Dictyostelium genes. The protein is extremely hydrophilic with a hydrophilic amino acid content of nearly 80%. The SQ1 protein contains a high content of glutamine (22%), asparagine (17%), and serine (9%). The asparagine and serine residues tend to be clustered in homopolymeric tracts and the glutamine residues are found primarily either in homopolymer tracts or in a repeated pentapeptide sequence. The pentapeptide sequence Gln-Glu-Gln-Leu-Lys is repeated nearly perfectly 37 times to form the central core of the protein. A search of Genbank and EMBL databases for similar proteins failed to reveal any highly related sequences. There was, however, some similarity between the pentapeptide repeat of SQ1 and a decapeptide repeat found in involucrin genes. Involucrin is the major keratinocyte structural protein that is apparently found only in primates. Although the function of the SQ1 gene cannot be unambiguously deduced from its predicted amino acid sequence, two possibilities can be suggested. The low levels of transcript and the high content of clustered glutamine residues are reminiscent of a number of transcription factors. However, no DNA binding domain was detected in the available SQ1 sequence. Alternatively, the pentapeptide repeats of SQ1 bear similarity to repeating sequences found in a number of structural and extracellular matrix proteins, including those of Dictyostelium.

Download

Share

COinS

Copyright for this work is retained by the student. If you have any questions regarding the inclusion of this work in the Digital Commons, please email us at .