BackgroundMutagenesis of yeast artificial chromosomes (YACs) often requires analysis of large numbers of yeast clones to obtain correctly targeted mutants. Conventional ways to isolate yeast genomic DNA utilize either glass beads or enzymatic digestion to disrupt yeast cell wall. Using small glass beads is messy, whereas enzymatic digestion of the cells is expensive when many samples need to be analyzed. We sought to develop an easier and faster protocol than the existing methods for obtaining yeast genomic DNA from liquid cultures or colonies on plates.ResultsRepeated freeze-thawing of cells in a lysis buffer was used to disrupt the cells and release genomic DNA. Cell lysis was followed by extraction with chloroform and ethanol precipitation of DNA. Two hundred ng – 3 μg of genomic DNA could be isolated from a 1.5 ml overnight liquid culture or from a large colony. Samples were either resuspended directly in a restriction enzyme/RNase coctail mixture for Southern blot hybridization or used for several PCR reactions. We demonstrated the utility of this method by showing an analysis of yeast clones containing a mutagenized human β-globin locus YAC.ConclusionAn efficient, inexpensive method for obtaining yeast genomic DNA from liquid cultures or directly from colonies was developed. This protocol circumvents the use of enzymes or glass beads, and therefore is cheaper and easier to perform when processing large numbers of samples.
The human β-globin locus is a complex genetic system widely used for analysis of eukaryotic gene expression. The locus consists of five functional β-like globin genes, ε, Gγ, Aγ, δ, and β, arrayed on the chromosome in the order that they are expressed during ontogeny. Globin gene expression is regulated, in part, by the locus control region, which physically consists of five DNasel-hypersensitive sites located 6-22 Kb upstream of the ε-globin gene. During ontogeny two switches occur in β-globin gene expression that reflect the changing oxygen requirements of the fetus. The first switch from embryonic ε- to fetal γ-globin occurs at six weeks of gestation. The second switch from γ- to adult δ- and β-globin occurs shortly after birth. Throughout the locus, cis-acting elements exist that are dynamically bound by trans-acting proteins, including transcription factors, co-activators, repressors, and chromatin modifiers. Discovery of novel erythroid-specific transcription factors and a role for chromatin structure in gene expression have enhanced our understanding of the mechanism of globin gene switching. However, the hierarchy of events regulating gene expression during development, from extracellular signaling to transcriptional activation or repression, is complex. In this review we attempt to unify the current knowledge regarding the interplay of cis-acting elements, transcription factors, and chromatin modifiers into a comprehensive overview of globin gene switching.
The eukaryotic cell cycle is regulated by cyclin-dependent kinases (CDKs). CDK4 and CDK6, which are activated by D-type cyclins during the G 1 phase of the cell cycle, are thought to be responsible for phosphorylation of the retinoblastoma gene product (pRb). The tumor suppressor p16INK4A inhibits phosphorylation of pRb by CDK4 and CDK6 and can thereby block cell cycle progression at the G 1 /S boundary. Phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II by general transcription factor TFIIH is believed to be an important regulatory event in transcription. TFIIH contains a CDK7 kinase subunit and phosphorylates the CTD. We have previously shown that p16INK4A inhibits phosphorylation of the CTD by TFIIH. Here we report that the ability of p16INK4A to inhibit CDK7-CTD kinase contributes to the capacity to induce cell cycle arrest. These results suggest that p16INK4A may regulate cell cycle progression by inhibiting not only CDK4-pRb kinase activity but also by modulating CDK7-CTD kinase activity. Regulation of CDK7-CTD kinase activity by p16INK4A thus may represent an alternative pathway for controlling cell cycle progression.Cyclin-dependent kinases (CDKs) regulate cell cycle progression (references 13, 21, and 28) and references therein). CDK4 and CDK6 are activated by D-type cyclins and participate in controlling the G 1 -to-S phase transition by phosphorylating the retinoblastoma gene product (pRb). Phosphorylation of pRb induces remodeling of transcriptional repressor complexes at pRb-regulated genes and causes the release of transcription factors such as E2F. Free E2F can then activate the transcription of genes required for entering S phase (36,41). p16 INK4A is a tumor suppressor gene product which binds CDK4 and inhibits CDK4-mediated phosphorylation of pRb (27). Overexpression of p16INK4A can block cell cycle progression through the G 1 -to-S phase boundary in a pRB-dependent manner (16,19). Many p16 INK4A mutants identified from human tumors have been shown to have defects in this activity (15,16,19,20,22,31). These data suggest that the CDK4-inhibitory activity of p16 INK4A is involved in regulating cell cycle progression through the G 1 /S boundary.Koh et al. have described an interesting phenotype associated with a p16INK4A mutant, G101W, that was originally identified in a familial melanoma kindred (14, 16). The G101W mutant was defective in inhibiting CDK4, although overexpression of the G101W mutant in an osteosarcoma cell line provoked cell cycle arrest at G 1 . In this mutant, the CDK4-pRb kinase-inhibitory activity of p16 INK4A apparently does not correlate with the ability to induce cell cycle arrest in G 1 when overexpressed. These results raise the possibility that an additional biochemical activity of p16INK4A might contribute to the ability to arrest cell cycle progression. (15,16,19,20,22,31,38,39). These data suggest that the ability to inhibit pRb kinase activity may not be the sole determinant of the tumor suppressor activity of p16 INK4A ....
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