Andrew W. Murray

Coding-sequence determinants of gene expression in Escherichia coli.

Synonymous mutations do not alter the encoded protein, but they can influence gene expression. To investigate how, we engineered a synthetic library of 154 genes that varied randomly at synonymous sites, but all encoded the same green fluorescent protein (GFP). When expressed in Escherichia coli, GFP protein levels varied 250-fold across the library. GFP messenger RNA (mRNA) levels, mRNA degradation patterns, and bacterial growth rates also varied, but codon bias did not correlate with gene expression. Rather, the stability of mRNA folding near the ribosomal binding site explained more than half the variation in protein levels. In our analysis, mRNA folding and associated rates of translation initiation play a predominant role in shaping expression levels of individual genes, whereas codon bias influences global translation efficiency and cellular fitness.

Recycling the cell cycle: cyclins revisited.

I discuss advances in the cell cycle in the 21 years since cyclin was discovered. The surprising redundancy amongst the classical cyclins (A, B, and E) and cyclin-dependent kinases (Cdk1 and Cdk2) show that the important differences between these proteins are when and where they are expressed rather than the proteins they phosphorylate. Although the broad principles of the cell cycle oscillator are widely accepted, we are surprisingly ignorant of its detailed mechanism. This is especially true of the anaphase promoting complex (APC), the machine that triggers chromosome segregation and the exit of mitosis by targeting securin and mitotic cyclins for destruction. I discuss how a cyclin/Cdk-based engine could have evolved to assume control of the cell cycle from other, older protein kinases.

Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores

The spindle assembly checkpoint delays anaphase until all chromosomes are attached to a mitotic spindle. The mad (mitotic arrest-deficient) and bub (budding uninhibited by benzimidazole) mutants of budding yeast lack this checkpoint and fail to arrest the cell cycle when microtubules are depolymerized. A frog homolog of MAD2 (XMAD2) was isolated and found to play an essential role in the spindle assembly checkpoint in frog egg extracts. XMAD2 protein associated with unattached kinetochores in prometaphase and in nocodazole-treated cells and disappeared from kinetochores at metaphase in untreated cells, suggesting that XMAD2 plays a role in the activation of the checkpoint by unattached kinetochores. This study furthers understanding of the mechanism of cell cycle checkpoints in metazoa and provides a marker for studying the role of the spindle assembly checkpoint in the genetic instability of tumors.

Chapter 30 Cell Cycle Extracts

Publisher Summary This chapter reviews the early Xenopus cycle and the history of cell cycle extracts. The early Xenopus cell cycles are controlled by the activation and inactivation of a protein kinase named maturation-promoting factor (MPF). MPF is also known as the cdc2 kinase and growth-associated histone H1 kinase, and consists of two principal subunits, the catalytic p34 cdc2 subunit and cyclin. Activation of MPF induces both mitosis and meiosis, whereas its inactivation triggers the onset of anaphase and the progression into interphase. The fluctuation of MPF activity in early Xenopus embryos is discussed in the chapter. The chapter discusses the methods for preparing and utilizing frog cell cycle extracts. Female frogs are primed for ovulation by injecting them with pregnant mare serum gonadotropin (PMSG) and then induced to ovulate by a subsequent injection of human chorionic gonadotropin (HCG). The chapter also describes sperm nucleus preparation.

Interphase chromosomes undergo constrained diffusional motion in living cells

BACKGROUND Structural studies of fixed cells have revealed that interphase chromosomes are highly organized into specific arrangements in the nucleus, and have led to a picture of the nucleus as a static structure with immobile chromosomes held in fixed positions, an impression apparently confirmed by recent photobleaching studies. Functional studies of chromosome behavior, however, suggest that many essential processes, such as recombination, require interphase chromosomes to move around within the nucleus. RESULTS To reconcile these contradictory views, we exploited methods for tagging specific chromosome sites in living cells of Saccharomyces cerevisiae with green fluorescent protein and in Drosophila melanogaster with fluorescently labeled topoisomerase ll. Combining these techniques with submicrometer single-particle tracking, we directly measured the motion of interphase chromatin, at high resolution and in three dimensions. We found that chromatin does indeed undergo significant diffusive motion within the nucleus, but this motion is constrained such that a given chromatin segment is free to move within only a limited subregion of the nucleus. Chromatin diffusion was found to be insensitive to metabolic inhibitors, suggesting that it results from classical Brownian motion rather than from active motility. Nocodazole greatly reduced chromatin confinement, suggesting a role for the cytoskeleton in the maintenance of nuclear architecture. CONCLUSIONS We conclude that chromatin is free to undergo substantial Brownian motion, but that a given chromatin segment is confined to a subregion of the nucleus. This constrained diffusion is consistent with a highly defined nuclear architecture, but also allows enough motion for processes requiring chromosome motility to take place. These results lead to a model for the regulation of chromosome interactions by nuclear architecture.

GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion.

BACKGROUND Precise control of sister chromatid separation is essential for the accurate transmission of genetic information. Sister chromatids must remain linked to each other from the time of DNA replication until the onset of chromosome segregation, when the linkage must be promptly dissolved. Recent studies suggest that the machinery that is responsible for the destruction of mitotic cyclins also degrades proteins that play a role in maintaining sister chromatid linkage, and that this machinery is regulated by the spindle-assembly checkpoint. Studies on these problems in budding yeast are hampered by the inability to resolve its chromosomes by light or electron microscopy. RESULTS We have developed a novel method for visualizing specific DNA sequences in fixed and living budding yeast cells. A tandem array of 256 copies of the Lac operator is integrated at the desired site in the genome and detected by the binding of a green fluorescent protein (GFP)-Lac repressor fusion expressed from the HIS3 promoter. Using this method, we show that sister chromatid segregation precedes the destruction of cyclin B. In mad or bub cells, which lack the spindle-assembly checkpoint, sister chromatid separation can occur in the absence of microtubules. The expression of a tetramerizing form of the GFP-Lac repressor, which can bind Lac operators on two different DNA molecules, can hold sister chromatids together under conditions in which they would normally separate. CONCLUSIONS We conclude that sister chromatid separation in budding yeast can occur in the absence of microtubule-dependent forces, and that protein complexes that can bind two different DNA molecules are capable of holding sister chromatids together.

Anaphase Is Initiated by Proteolysis Rather Than by the Inactivation of Maturation-Promoting Factor

We have used frog egg extracts that assemble mitotic spindles to identify the event that triggers sister chromatid separation. Adding a nondegradable form of cyclin B prevents maturation-promoting factor (MPF) inactivation but does not block sister chromatid separation, showing that MPF inactivation is not needed to initiate anaphase. In contrast, adding an N-terminal fragment of cyclin, which acts as a specific competitor for cyclin degradation, produces a dose-dependent delay in MPF inactivation and sister chromatid separation. Methylated ubiquitin, which inhibits ubiquitin-mediated proteolysis, also delays sister chromatid separation, suggesting that ubiquitin-mediated proteolysis is necessary to initiate anaphase. The N-terminal cyclin fragment inhibits chromosome separation even in extracts that contain only nondegradable forms of cyclin, suggesting that proteins other than the known cyclins must be degraded to dissolve the linkage between sister chromatids.

Chromosome and Low Copy Plasmid Segregation in E. coli: Visual Evidence for Distinct Mechanisms

We have investigated DNA segregation in E. coli by inserting multiple lac operator sequences into the chromosome near the origin of replication (oriC), in the hisC gene, a terminus marker, and into plasmids P1 and F. Expression of a GFP-LacI fusion protein allowed visualization of lac operator localization. oriC was shown to be specifically localized at or near the cell poles, and when duplicated, one copy moved to the site of new pole formation near the site of cell division. In contrast, P1 and F localized to the cell center and on duplication appeared to move rapidly to the quarter positions in the cell. Our analysis suggests that different active processes are involved in movement and localization of the chromosome and of the two plasmids during segregation.

A MAP kinase-dependent spindle assembly checkpoint in Xenopus egg extracts.

Like early Xenopus embryos, extracts made from Xenopus eggs lack the cell cycle checkpoint that keeps anaphase from occurring before spindle assembly is complete. At very high densities of sperm nuclei, however, microtubule depolymerization arrests the extracts in mitosis. The arrested extracts have high levels of maturation-promoting factor activity, fail to degrade cyclin B, and contain activated ERK2/mitogen-activated protein (MAP) kinase. The addition of the purified MAP kinase-specific phosphatase MKP-1 demonstrates that MAP kinase activity is required for both the establishment and maintenance of the mitotic arrest induced by spindle depolymerization. Increased calcium concentrations, which release unfertilized frog eggs from their natural arrest in metaphase of meiosis II, have no effect on the mitotic arrest.

The spindle assembly checkpoint.

The spindle assembly checkpoint monitors proper chromosome attachment to spindle microtubules and is conserved from yeast to humans. Checkpoint components reside on kinetochores of chromosomes and show changes in phosphorylation and localization as cells proceed through mitosis. Adaptation to prolonged checkpoint arrest can occur by inhibitory phosphorylation of Cdc2.