Paul Jorgensen

How cells coordinate growth and division.

Size is a fundamental attribute impacting cellular design, fitness, and function. Size homeostasis requires a doubling of cell mass with each division. In yeast, division is delayed until a critical size has been achieved. In metazoans, cell cycles can be actively coupled to growth, but in certain cell types extracellular signals may independently induce growth and division. Despite a long history of study, the fascinating mechanisms that control cell size have resisted molecular genetic insight. Recently, genetic screens in Drosophila and functional genomics approaches in yeast have macheted into the thicket of cell size control.

Proteolysis and the cell cycle: with this RING I do thee destroy.

The ubiquitin system drives the cell division cycle by the timely destruction of numerous regulatory proteins. Remarkably, the two main activities that catalyze substrate ubiquitination in the cell cycle, the Skp1-Cdc53/cullin-F-box protein (SCF) complexes and the anaphase-promoting complex/cyclosome (APC/C), define a new superfamily of E3 ubiquitin ligases, all based on related cullin and RING-H2 finger protein subunits. The circuits that interconnect the SCF, APC/C and cyclin-dependent kinase activities form a master oscillator that coordinates the replication and segregation of the genome.

Dissection of combinatorial control by the Met4 transcriptional complex.

Loss of Met31 and Met32 abolishes all Met4-activated transcription, while only certain target genes, such as sulfate assimilation genes, depend on Cbf1 and Met28 for expression. Unlike Met4 and the other cofactors, Cbf1 remains promoter-bound under inducing and repressing conditions and helps to stabilize Met32, the main platform for Met4, at promoters.

The fork'ed path to mitosis

A concurrence of genomic, reverse genetic and biochemical approaches has cracked the decade-long enigma concerning the identity of the transcription factors that control gene expression at the G2/M transition in the budding yeast cell cycle.

AFM 4.0: a toolbox for DNA microarray analysis

We have developed a series of programs, collectively packaged as Array File Maker 4.0 (AFM), that manipulate and manage DNA microarray data. AFM 4.0 is simple to use, applicable to any organism or microarray, and operates within the familiar confines of Microsoft Excel. Given a database of expression ratios, AFM 4.0 generates input files for clustering, helps prepare colored figures and Venn diagrams, and can uncover aneuploidy in yeast microarray data. AFM 4.0 should be especially useful to laboratories that do not have access to specialized commercial or in-house software.

Systematic Validation and Atomic Force Microscopy of Non-Covalent Short Oligonucleotide Barcode Microarrays

Background Molecular barcode arrays provide a powerful means to analyze cellular phenotypes in parallel through detection of short (20–60 base) unique sequence tags, or “barcodes”, associated with each strain or clone in a collection. However, costs of current methods for microarray construction, whether by in situ oligonucleotide synthesis or ex situ coupling of modified oligonucleotides to the slide surface are often prohibitive to large-scale analyses. Methodology/Principal Findings Here we demonstrate that unmodified 20mer oligonucleotide probes printed on conventional surfaces show comparable hybridization signals to covalently linked 5′-amino-modified probes. As a test case, we undertook systematic cell size analysis of the budding yeast Saccharomyces cerevisiae genome-wide deletion collection by size separation of the deletion pool followed by determination of strain abundance in size fractions by barcode arrays. We demonstrate that the properties of a 13K unique feature spotted 20 mer oligonucleotide barcode microarray compare favorably with an analogous covalently-linked oligonucleotide array. Further, cell size profiles obtained with the size selection/barcode array approach recapitulate previous cell size measurements of individual deletion strains. Finally, through atomic force microscopy (AFM), we characterize the mechanism of hybridization to unmodified barcode probes on the slide surface. Conclusions/Significance These studies push the lower limit of probe size in genome-scale unmodified oligonucleotide microarray construction and demonstrate a versatile, cost-effective and reliable method for molecular barcode analysis.

Altered states: programmed proteolysis and the budding yeast cell cycle.

The recent identification of an essential RING-H2 finger protein in the SCF E3 ubiquitin ligase complex of budding yeast has uncovered a family of related E3 enzymes, including the other main cell cycle E3 complex, the anaphase promoting complex (APC). Recent insights into APC-dependent proteolysis include a novel protease activity that dissolves cohesion between sister chromatids at anaphase, and a crucial phosphatase, Cdc14, whose release from the nucleolus eliminates cyclin-dependent kinase activity and thereby drives exit from mitosis.

Microarray Analysis of Genes Induced by Methionine Starvation and Growth on Different Sulfur Sources in Yeast

Methionine is an important end-product of folate metabolism and it is reasonable to expect that the expression of several folate-dependent enzymes might be regulated by its availability. In the yeast, Saccharomyces cerevisiae, previous studies have shown that at least 20 genes of sulfate reduction and methionine biosynthesis are regulated by the transcription factor Met4p in response to methionine, cysteine or S-adenosylmethionine levels (1). Met4p lacks a DNA-binding domain and requires either Cbflp or the redundant factors Met31p or Met32p to bind to DNA at the promoters it regulates. Cbflp binds to the consensus sequence TCACGTG, while Met31 and Met32p both bind to the consensus sequence AAACTGTG. Another factor, Met28p increases the affinity of Cbflp binding and is also present in the Met4p-Met31p/Met32p promoter complex (2). Met4p is degraded or inhibited in response to excess methionine following ubiquitination by the SCFMET30complex (3, 4).