Functional analysis of Cdk7-interacting proteins Mat1 and Hint in model organisms

............................................................................................................................................7 REVIEW OF THE LITERATURE ......................................................................................................9 1 THE CELL DIVISION CYCLE.................................................................................................................9 1.1 Cyclin-dependent kinases (Cdks) ..............................................................................................9 1.2 Regulation of Cdks ..................................................................................................................10 2 CDK-ACTIVATING KINASES..............................................................................................................11 2.1 Ménage à trois (MAT1)...........................................................................................................12 2.2 The Cdk-activating kinase – from mammals to yeast..............................................................13 3 CDK ACTIVATION MEETS BASAL TRANSCRIPTION ............................................................................13 3.1 Transcription factor IIH (TFIIH)............................................................................................14 3.2 Substrates of the TFIIH kinase................................................................................................15 4 HISTIDINE TRIAD (HIT) GENE FAMILY ............................................................................................17 4.1 The origin of Hint....................................................................................................................18 4.2 FHIT........................................................................................................................................18 4.3 GalT.........................................................................................................................................19 4.4 Novel members of the HIT family: aprataxin, Hint2, and Hint3 ............................................19 4.5 HIT proteins – from structure to function...............................................................................20 4.6 Other Hint-interacting cellular proteins.................................................................................22 AIMS OF THE STUDY .......................................................................................................................23 MATERIALS AND METHODS.........................................................................................................24 Generation of genetically engineered Mat1 (I, II) and Hint mice (IV).....................................................24 Generation and analysis of Hint;Fhit mice (IV)...................................................................................24 Generation of S. cerevisiae HNT1 disruptant (III) ....................................................................................24 PCR genotyping (I, II, III, IV) ..................................................................................................................25 Protein analysis (I, II, III, IV) ...................................................................................................................26 Immunofluorescence and immunohistochemistry (I, II, III)..................................................................... 26 BrdU labeling (I, II) ..................................................................................................................................26 Blastocyst outgrowths (I) .........................................................................................................................27 Microinjection (I)......................................................................................................................................27 Histology (II, IV) ......................................................................................................................................27 Transmission electron microscopy (II) .....................................................................................................27 ß-galactosidase and alkaline phosphatase staining (II) .............................................................................27 Yeast strains and culture conditions (III)..................................................................................................28 Yeast transformation (III) .........................................................................................................................28 Production of GST proteins and solution binding assay (III) ...................................................................28 Transfection (III).......................................................................................................................................29 In vitro kinase assay (III, IV) ....................................................................................................................29 In situ hybridization (IV) ..........................................................................................................................29 Modeling of three dimensional structure (IV) ..........................................................................................30