Functional analysis of the mouse G90 gene

Meunier, Dominique

Local TagsRelease HistoryDetailsSummary

Functional analysis of the mouse G90 gene

Meunier, D. (2003). Functional analysis of the mouse G90 gene. PhD Thesis, Albert-Ludwigs-Universität, Freiburg im Breisgau.

Item is Released

show all hide all

Basic

show hide

Item Permalink: https://hdl.handle.net/11858/00-001M-0000-0010-8B4D-B Version Permalink: https://hdl.handle.net/11858/00-001M-0000-0010-8B4E-9

Genre: Thesis

Files

show Files

hide Files

:

PhD-Meunier.pdf (Any fulltext), 5MB

File Permalink:
-

Name:
PhD-Meunier.pdf

Description:
-

OA-Status:

Visibility:
Restricted (Max Planck Institute for Molecular Genetics, MBMG; )

MIME-Type / Checksum:
application/pdf

Technical Metadata:

Copyright Date:
-

Copyright Info:
eDoc_access: MPG

License:
-

Locators

show

Creators

show

hide

Creators:
Meunier, Dominique¹, Author

Affiliations:
1Max Planck Society, ou_persistent13

Content

show

hide

Free keywords: -

Abstract: The G90 gene was isolated a few years ago in a screen aimed at identifying genes expressed differentially between the small and large intestines of adult mice. This novel mouse gene shows no homology to known genes or gene families and its function is as yet unknown. However, G90 exhibits several interesting characteristics, including an intriguing mouse strain-dependent allelic usage and a distinct expression pattern in embryonic and adult mouse tissues, as well as in mouse intestinal tumours. It has previously been shown that G90 expression is restricted to post-mitotic cells in the intestine and testis of adult mice, thus suggesting a role in the control of proliferation and/or differentiation. In an attempt to get an insight into the possible function of G90, a detailed characterization of this gene was undertaken. First, RT-PCR and RNA FISH analyses revealed that G90 is monoallelically expressed in the majority of adult mouse intestinal cells; the mechanisms underlying the allelic expression of this gene remain however unknown. Then, database analyses led to the discovery that G90, which was first described as a noncoding RNA, in fact encodes a short protein conserved throughout mammalian and vertebrate evolution, a finding that strongly supports the view that this novel gene is functional. The detailed analysis of the spatiotemporal expression pattern of G90 during mouse embryonic development supported the hypothesis that this gene might be involved in the control of proliferation or differentiation: the gene was found to be expressed at high levels in specific structures of the developing head and a clear correlation between G90 expression and absence of proliferation could be observed in most of these tissues. However, the analysis of transgenic mice expressing the human G90 gene, as well as the use of various candidate gene and and microarray analysis approaches, failed to establish a link between G90 and a number of important regulators of the cell cycle, including the cyclin-dependent kinase inhibitors CDKN1A and CDKN1B, and the tumour suppressor TP53. Furthermore, the expression of the human G90 gene in embryos and mice did not cause any obvious phenotype, thus bringing no conclusive evidence as to the possible function of G90. However, the G90 gene was found to be specifically expressed in gastrointestinal tumours derived from Apc-deficient mice, as well as in several human colorectal cancers. Interestingly, its expression in such tumours appeared to correlate with defects in the Wnt signalling pathway. These findings suggest that G90 might play a role in the development or progression of gastrointestinal tumours. Thus, although the function of the G90 gene could not be elucidated in the course of the present study, the results obtained clearly indicate that this gene is likely to be functional. Additional studies should help to clarify the putative function of G90 in proliferation, differentiation, tumorigenesis and other cellular processes.

Details

show

hide

Language(s): eng - English

Dates: Accepted: 2003

Publication Status: Accepted / In Press

Pages: -

Publishing info: Freiburg im Breisgau : Albert-Ludwigs-Universität

Table of Contents: Abstract ........................... III
Acknowledgements ................... V
Table of contents .................. VI
List of figures .................... X
List of tables ..................... XI
List of abbreviations ...............XII
Chapter 1 - Introduction.............. 1
1.1 The G90 gene ..................... 1
1.1.1 Organization of the G90 gene ... 1
1.1.2 Allelic expression of the G90 gene in adult mouse intestinal cells ..................... 2
1.1.3 Expression of the G90 gene in mouse tissues ...................................... 3
1.1.4 Hypotheses regarding the function of the G90 gene ...................................... 3
1.2 Regulation of cell cycle progression, proliferation and differentiation ...................... 6
1.2.1 Regulation of cell cycle progression ...................................... 6
1.2.1.1 Cyclins and cyclin-dependent kinases ...................................... 6
1.2.1.2 Cyclin-dependent kinase inhibitors and retinoblastoma transcriptional repressors ...................................... 7
1.2.1.3 TP53, the gatekeeper of genomic integrity ...................................... 8
1.2.2 Coordination of proliferation and differentiation ...................................... 9
1.3 The mammalian intestinal tract... 12
1.3.1 Structure and function of the intestinal tract ..................................... 12
1.3.2 The crypt-villus axis: physiology and self-renewal ..................................... 12
1.3.3 Pathological conditions ....... 14
1.4 Colorectal cancer ............... 14
1.4.1 The colorectal adenoma-carcinoma sequence ..................................... 14
1.4.2 Genes and pathways involved in colorectal cancer ..................................... 15
1.4.2.1 Wnt signalling pathway and the APC protein ..................................... 15
1.4.2.2 TGF-b signalling pathway and the SMAD proteins ..................................... 15
1.4.2.3 The tumour suppressor TP53 .. 16
1.4.2.4 Cyclin-dependent kinase inhibitors ..................................... 16
1.5 Aims of the G90 project.......... 17
Chapter 2 - Materials and Methods ... 18
2.1 Standard solutions .............. 18
2.2 Plasmids ........................ 18
2.3 Animal and human materials ...... 21
2.3.1 Mouse strains ................. 21
2.3.2 Generation of human G90 transgenic mice ..................................... 22
2.3.3 Generation of homozygous hG90 transgenic mice ..................................... 22
2.3.4 Generation of hG90tg/ApcMin/+ compound mice ..................................... 22
2.3.5 Cattle ........................ 22
2.3.6 Zebrafish ..................... 23
2.3.7 Human material ................ 23
2.4 Tissue isolation and processing . 23
2.4.1 Embryonic and adult mouse tissues ..................................... 23
2.4.2 Cattle, zebrafish and human tissues ......................................23
2.5 Genotyping ...................... 24
2.5.1 Genomic DNA isolation ......... 24
2.5.2 Genotyping by PCR ............. 24
2.5.2.1 PCR primers ................. 24
2.5.2.2 PCR reactions ............... 25
2.5.3 Genotyping by Southern blot ... 25
2.5.3.1 Radioactive probe labelling . 25
2.5.3.2 Southern blot ................26
2.5.4 Genotyping by chromosomal fluorescent in situ hybridization (FISH) ................ 26
2.5.4.1 Fibroblast culture .......... 26
2.5.4.2 Chromosome isolation from fibroblasts ..................................... 27
2.5.4.3 Chromosomal FISH ............ 27
2.5.4.3.1 Probe labelling by nick translation ..................................... 27
2.5.4.3.2 Chromosomal FISH .......... 28
2.6 RNA isolation, cDNA synthesis and RT-PCR ..................................... 28
2.6.1 RNA isolation ................. 28
2.6.2 cDNA synthesis ................ 28
2.6.3 RT-PCR ........................ 29
2.6.3.1 PCR primers ................. 29
2.6.3.2 RT-PCR reactions ............ 29
2.7 Cloning and plasmid DNA preparation ..................................... 30
2.7.1 PCR and RT-PCR product purification ..................................... 30
2.7.2 Ligation and transformation ... 30
2.7.3 Plasmid DNA purification........31
2.8 DNA sequencing .................. 31
2.8.1 Sequencing with the Li-Cor DNA sequencer ..................................... 31
2.8.2 Sequencing with the ABI PRISM sequencer ..................................... 31
2.9 Northern blot analyses .......... 32
2.9.1 DNA fragments for probe labelling ..................................... 32
2.9.2 Radioactive probe labelling ... 32
2.9.3 Northern blot.................. 32
2.9.4 Normalisation and quantification of RNA levels ..................................... 32
2.10 Histological analyses .......... 33
2.10.1 Hematoxylin/Eosin (HE) staining ..................................... 33
2.10.2 Periodic acid Schiff (PAS) staining ..................................... 33
2.10.3 Alcian blue staining ......... 33
2.10.4 Combined alcian blue / PAS staining ..................................... 34
2.11 Immunohistological analyses .... 34
2.11.1 Primary antibodies ........... 34
2.11.2 Immunostaining reactions ..... 34
2.12 Radioactive RNA in situ hybridization (ISH) ..................................... 35
2.12.1 Plasmids for riboprobe in vitro transcription ..................................... 35
2.12.2 Riboprobe in vitro transcription ..................................... 35
2.12.3 RNA ISH ...................... 36
2.13 RNA fluorescent in situ hybridization (FISH) ..................................... 36
2.13.1 Preparation of the samples ... 36
2.13.2 Probe labelling by nick translation ..................................... 37
2.13.3 RNA FISH...................... 37
2.14 Microarray analyses ............ 37
2.14.1 Fluorescent cDNA labelling ... 37
2.14.2 Microarray hybridization ..... 38
2.14.3 Image acquisition and data analysis ..................................... 38
2.15 Radiation hybrid mapping ....... 38
2.16 5-fluorouracil experiment ...... 38
2.17 Grading of mouse and human intestinal tumours ..................................... 39
2.18 Sequence and database analyses ..................................... 39
Chapter 3 - Results ................. 40
3.1 Allelic expression of the G90 gene ..................................... 40
3.2 Conservation of G90 during vertebrate evolution ..................................... 43
3.2.1 Conservation of G90 at the cDNA and protein levels ..................................... 43
3.2.2 Chromosomal mapping of G90 orthologs ..................................... 45
3.2.3 Conservation of G90 expression pattern ..................................... 46
3.3 Expression of G90 during mouse embryonic and post-natal development ......................... 51
3.3.1 Expression of G90 in the developing head ..................................... 51
3.3.2 Expression of G90 in the developing inner ear ..................................... 52
3.3.3 Expression of G90 in non-proliferating cells of the developing head ..................... 57
3.4 Human G90 transgenic mice ....... 60
3.4.1 Analysis of hG90tg adult mice ..60
3.4.2 Analysis of hG90tg embryos .... 61
3.4.3 Analysis of homozygous hG90tg embryos and mice ..................................... 65
3.5 Candidate gene approaches: seeking the function of G90 ...........................,,,,...... 68
3.5.1 G90 and Cdkn1b do not interact during inner ear development ......................... 68
3.5.2 G90 does not interact with Cdkn1a, Ckdn1b, Rbl1 and Rbl2 in mouse intestinal epithelium . 71
3.5.3 G90 expression is not affected by TP53-dependent intestinal apoptosis induced by 5-fluorouracil ............................. ....... 71
3.6 Microarray analyses: seeking G90 target genes ..................................... 73
3.7 Expression of G90 in mouse and human GI tumours ..................................... 75
3.7.1 G90 expression in GI tumours from Apc mouse models ..................................... 75
3.7.2 G90 expression in human GI tumours ..................................... 76
3.7.3 G90 is not expressed in GI tumours from non-Apc mouse models of CRC ....................... 81
3.7.4 Expression of the hG90 transgene does not affect tumour formation in ApcMin/+ mice ... 83
3.7.5 Up-regulation of hG90 expression in growth-arrested CRC cells ........................... 84
Chapter 4 - Discussion .............. 88
4.1 Predominant monoallelic expression of G90 in adult mouse intestinal cells .................... 88
4.2 G90 is likely to encode a protein conserved throughout vertebrate evolution ................ 89
4.3 The spatio-temporal expression pattern of G90 during mouse embryonic development supports a role in proliferation or differentiation .................. 92
4.4 Seeking the function of the G90 gene ..................................... 95
4.5 The G90 gene appears to be a target of the Wnt signalling pathway in GI tumours .... 97
4.6 The hG90tg mice: an appropriate mouse model? ..................................... 99
List of references.................. 102

Rev. Type: -

Identifiers: eDoc: 194785

Degree: PhD

Event

show

Legal Case

show

Project information

show

Source

show