1: Mol Cell Biochem. 1999 Dec;202(1-2):109-18. 

Characterization of Saccharomyces cerevisiae strains expressing ira1 mutant
alleles modeled after disease-causing mutations in NF1.

Gil R, Seeling JM.

Department of Oncological Sciences and Huntsman Cancer Institute, University of
Utah, Salt Lake City, USA.

The 2818 amino acids of neurofibromin, the product of the human NF1 gene,
include a 230 amino acid Ras-GAP related domain (GRD). Functions which may be
associated with the rest of the protein remain unknown. However, many NF1
mutations in neurofibromatosis 1 patients are found downstream of the GRD,
suggesting that the C-terminal region of the protein is also functionally
important. Since the C-terminal region of neurofibromin encompassing these
mutations is homologous with the corresponding regions in the two Saccharomyces
cerevisiae Ras-GAPs, Ira1p and Ira2p, we chose yeast as a model system for
functional exploration of this region (Ira-C region). Three missense mutations
that affect the Ira-C region of NF1 were used as a model for the mutagenesis of
IRA1. The yeast phenotypes of heat shock sensitivity, iodine staining,
sporulation efficiency, pseudohyphae formation, and GAP activity were scored.
Even though none of the mutations directly affected the Ira1p-GRD, mutations at
two of the three sites resulted in a decrease in the GAP activity present in
ira1 cells. The third mutation appeared to disassociate the phenotypes of
sporulation ability and GAP activity. This and other evidence suggest an
effector function for Ira1p.

PMID: 10706001 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


2: Endocrinology. 1996 Sep;137(9):3897-905. 

cis-acting elements and trans-acting proteins in the transcription of chorionic
gonadotropin/luteinizing hormone receptor gene in human choriocarcinoma cells
and placenta.

Hu YL, Lei ZM, Rao CV.

Department of Obstetrics and Gynecology, University of Louisville School of
Medicine, Kentucky 40292, USA.

We investigated the cis-acting elements and trans-acting proteins responsible
for a higher basal rate of transcription of hCG/LH receptor gene in human
choriocarcinoma JEG-3 cells compared with normal term pregnancy placenta.
Sequential deletion of the 5'-flanking region of the gene revealed that there
are three negative control regions (NCRs) designated NCR1 (-1457 to -1373 bp),
NCR2 (-1051 to -835 bp), and NCR3 (-480 to -184 bp), and a promoter (-184 to -1
bp). NCR3 was more inhibitory than the other two; nearly 60-70% of the
inhibitory activity resides in a sequence between -480 to -276 bp, and the rest
resides in the sequence between -276 to -184 bp. Gel mobility shift assays
showed that the nuclear extracts from JEG-3 cells contained proteins that form
three complexes with NCR1, two with NCR2, and six with NCR3. Many of the
proteins that form the complexes in NCR3 are shared with the other two NCRs.
Most of the proteins that form these complexes are less abundant in nuclear
extracts from JEG-3 cells than in those from placenta. The JEG-3 cell nuclear
extracts also contained proteins that form three complexes with the proximal
promoter of the hCG/LH receptor gene. These proteins were identified as Ap2,
Ap2-like I, and Sp1 from the competition studies with synthetic excess unlabeled
Ap2, Sp1, and CTF/NF1 consensus oligodeoxynucleotides and/or supershift in gel
mobility assays with anti-Ap2 antibody. Although the JEG-3 cell nuclear extracts
contained abundant Ap2-like protein I and low levels of Ap2 and Sp1 proteins,
the placental nuclear extracts contained low levels of Ap2-like protein I and
very low to nondetectable levels of Ap2 and Sp1 proteins. Deoxyribonuclease I
footprinting revealed that the nuclear extracts from JEG-3 cells and placent
protected the -116 to -93 bp and -65 to -45 bp regions in the proximal promoter
of the hCG/LH receptor gene that contain Sp1 and Ap2 binding sites,
respectively. However, the nuclear extracts from placenta only partially
protected these regions, which is consistent with lower levels of proteins that
bind to the proximal promoter of the gene. In summary, we conclude that the
presence of low levels of proteins that bind to the NCRs and the high levels of
proteins, especially Ap2-like I, that bind to the proximal promoter can
potentially explain higher transcription of the hCG/LH receptor gene in JEG-3
cells compared with that in normal term pregnancy human placenta.

PMID: 8756564 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


3: Cancer Genet Cytogenet. 1996 May;88(1):17-25. 

Cytogenetic and histologic correlation of peripheral nerve sheath tumors of soft
tissue.

Rao UN, Surti U, Hoffner L, Yaw K.

Department of Pathology, University of Pittsburgh Medical Center, Pennsylvania,
15213-2582, USA.

Cytogenetic analysis was performed on 11 peripheral nerve sheath tumors of soft
tissue from 10 patients. They include 6 benign and 5 malignant schwannomas. Five
cases which include two benign, one cellular and two malignant schwannomas had a
known association with a nerve, but only one patient with malignant schwannoma
has clinically documented neurofibromatosis type I. All the patients had a
normal diploid constitutional karyotype. Two cases of cellular schwannoma were
analyzed by routine cytogenetic analysis and fluorescence in situ hybridization
(FISH). One tumor was karyotyped as 45, XX,-13,-22 +mar; and the other case had
a 45,X,-Y,t(1;17) (p12;q11.2) karyotype. In the latter, the breakpoint in 17q
occurred below the centromere and is at or in the region of the
Neurofibromatosis Type 1 (NF1) gene. Four benign tumors had a normal diploid
karyotype. One hypodiploid malignant schwannoma with myxoid features
demonstrated monosomy of chromosomes 17 and 22 by FISH analysis. The rest of the
malignant schwannomas showed a wide range of numerical and structural
aberrations, with frequent loss of 22q and gains of chromosomes 2 and 7. Loss of
a sex chromosome was observed in cellular as well as malignant schwannomas.
Regional karyotypic evolution was noted in one malignant schwannoma. Cytogenetic
analysis may prove to be useful in identifying tumors, such as cellular
schwannomas, which, because of their histologic features may be inadvertently
categorized as malignant. Simultaneous involvement of NF1 and NF2 genes, which
are located on chromosomes 17q and 22q, respectively, should be investigated at
a molecular level in both benign and malignant tumors of peripheral nerves.

PMID: 8630973 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------