Staff
Group leader: GIORGIO SESTI
Universita' degli Studi di ROMA "Tor Vergata"
Medicina e Chirurgia
Affiation Dipartimento di medicina Interna
1. FEDERICI MASSIMO Università di Roma Tor Vergata Dipartimento di Medicina Interna
2. SANGIUOLO FEDERICA Università di Roma Tor Vergata Dipartimento di Biopatiologia e Diagnostica Per Immagini
The research line n.2 is focused to efficiently generate
and isolate high quality, full-length coding sequences from information contained
in Type 2 diabetes-specific expressed sequence tags (ESTs).
Generation of full-length cDNA clones is fundamental to provide accurate and
complete information for gene structure and functional analysis. Most of the
current approaches for mapping disease-susceptible genes are able to identify
and isolate only partial expressed sequences. The common subsequent step consisting
in the screening of a specific cDNA library is very tedious, laborious and often
not even sufficient to obtain full-length coding sequences. In fact, despite
the development of numerous strategies for constructing full-length cDNA libraries,
most of these libraries contain partial cDNA clones (0.5-2 kb) lacking sequences
at the 5'end. Rapid amplification of cDNA end (RACE) has been used to clone
full-length cDNAs. However, production of a full-length cDNA clone by RACE requires
substantial subcloning and ligation of different partial sequences. Thus, the
entire process of obtaining full-length cDNA clones is still highly inefficient
and time-consuming. An alternative strategy able of bypassing the library screening
step can be extremely useful also in the characterization of differentially
expressed genes. Methodologies that are commonly used for isolating unknown
differentially expressed genes only identify short cDNA fragments that need
to be fully characterized by the additional methods cited above.
The strategy proposed will use a novel library-free approach that enables rapid
isolation of distinct mRNA species and subsequent production of full-length
cDNA clones by a capture-primer enrichment technology (Patent pending). The
principle underlying this approach has been already validated using the human
actin genes as a model target cDNA (1,2).
Our analysis will start from specific type 2 diabetes ESTs that will be isolated
by differential display or PCR-based subtractive hybridization strategy (3)
from total cellular RNA extracted by fat and skeletal muscle biopsies obtained
from subphenotypes of the first-degree relatives (FDR) of patients with Type
II diabetes for the identification of specific genes that are dysregulated and
associated with risk for Type 2 diabetes.
Starting from these specific expressed sequence, full length cDNAs clones will
readily be cloned using this novel library-free system. The proposed method
is based on the enrichment for a gene or EST-specific mRNA prior to cDNA synthesis,
and as such would also insure representation of low abundance mRNA species,
while in conventional cDNA libraries, these rarely expressed mRNA occur at a
frequency of £ 10-6. The hypothesis that underlies this approach is that
full-length cDNA are more readily synthesized with relatively homogeneous, EST-specific
mRNAs than a heterogeneous population of poly(A)+ RNA. By hybridizing with EST-derived
biotin labeled antisense primers, EST-specific mRNAs will be captured and isolated
from total cellular RNA with streptavidin-coated magnetic beads. The enriched
and purified EST-specific mRNA will then be used for an improved version of
cDNA synthesis and cloned into expression plasmid vectors. Screening for desired
clones would be performed by multiplex primer-specific PCR. Since the cDNA clones
are derived from highly enriched mRNAs it is expected that screening for full-length
cDNA clones will be more efficient and more cost-effective than it would be
for screening a conventional cDNA library. Positive clones will be subjected
to automatic sequencing and gene structure characterisation. They will then
investigated to search causative SNPs as described in Research line 1.
1. Xu Z. and Gruenert, D.C. Development of a library-free
system for EST-specific full-length cDNA cloning. Genome Tri-conference, San
Francisco, CA (1998).
2. Xu Z., Jablons D.M., Gruenert D.C. EST-specific full-length cDNA clonong:
actin cDNAs. submitted (2000)
3. Zhu F., Yan W., Zhao Z.L., Chai Y.B. et al. Improved PCR-based subtractive
hybridization strategy for cloning differentially expressed genes. BioTechniques
29:310-313 (2000).
Amount (ML) 75
Source(s) Italian Telethon and Italian Ministry of Health
1) Hribal ML, Federici M, Porzio O, Lauro D, Borboni P, Accili D, Lauro R, Sesti
G The Gly-->Arg972 amino acid polymorphism in insulin receptor substrate-1
affects glucose metabolism in skeletal muscle cells. J Clin Endocrinol Metab
2000 May;85(5):2004-13
2) Federici M, Giaccari A, Hribal ML, Giovannone B, Lauro D, Morviducci L, Pastore L, Tamburrano G, Lauro R, Sesti G Evidence for glucose/hexosamine in vivo regulation of insulin/IGF-I hybrid receptor assembly. Diabetes 1999 Dec;48(12):2277-85
3) Porzio O, Federici M, Hribal ML, Lauro D, Accili D, Lauro R, Borboni P, Sesti G. The Gly972-->Arg amino acid polymorphism in IRS-1 impairs insulin secretion in pancreatic beta cells.J Clin Invest 1999 Aug;104(3):357-64.
4) Federici M, Lauro D, D'Adamo M, Giovannone B, Porzio O, Mellozzi M, Tamburrano G, Sbraccia P, Sesti G Expression of insulin/IGF-I hybrid receptors is increased in skeletal muscle of patients with chronic primary hyperinsulinemia. Diabetes 1998 Jan;47(1):87-92
5) Federici M, Zucaro L, Porzio O, Massoud R, Borboni P, Lauro D, Sesti G Increased expression of insulin/insulin-like growth factor-I hybrid receptors in skeletal muscle of noninsulin-dependent diabetes mellitus subjects. J Clin Invest 1996 Dec 15;98(12):2887-93