D J Saul — Page 2 | PubMed Health

PubMed1985

Molecular cloning of WHI2, a gene involved in the regulation of cell proliferation in Saccharomyces cerevisiae.

The WHI2 gene of Saccharomyces cerevisiae plays a key role in coordinating cell proliferation and nutrient availability. A 2.6 kb yeast DNA sequence has been cloned which fully suppresses the whi2 mutation. Integration of this sequence to demonstrate that the structural gene itself had been cloned proved difficult. Integration occurred only rarely and only at the LEU2 locus which was also present on the integrating plasmid. To circumvent these difficulties an adjacent sequence, present on the original isolate from the gene library, was subcloned onto the integrating vector YIp5, after which directed integration proved straightforward. The integrated sequence was closely linked to WHI2, confirming that the structural gene had been cloned. A chromosomal restriction map of the WHI2 region is presented; no gross changes were observed in the region as cells entered stationary phase.

Autoradiography↗

PubMedDate not supplied

celA, another gene coding for a multidomain cellulase from the extreme thermophile Caldocellum saccharolyticum.

Caldocellum saccharolyticum is an extremely thermophilic anaerobic bacterium capable of growth on cellulose and hemicellulose as sole carbon sources. Cellulase and hemicellulase genes have been found clustered together on its genome. The gene for one of the cellulases (celA) was isolated on a lambda genomic library clone, sequenced and found to comprise a large open-reading frame of 5253 base pairs that could be translated into a peptide of 1751 amino acids. To date, it is the largest cellulase gene sequenced. The translated product is a multidomain structure composed of two catalytic domains and two cellulose-binding domains linked by proline-threonine-rich regions (PT linkers). The N-terminal domain of celA encodes for an endoglucanase activity on carboxymethylcellulose, consistent with its high homology to the sequences of several other endo-1, 4-beta-D-glucanases. The carboxyterminal domain shows sequence homology with a cellulase from Clostridium thermocellum (CelS), which is known to act synergistically with a second component to hydrolyze crystalline cellulose. In the absence of a Caldocellum homologue for this second protein, we can detect no activity from this domain.

Amino Acid Sequence↗