High School Students Recruited
through the Karmanos Cancer Institute
Garrett Heffner (L) and Brandee Brewer (R) are high school
students from the Detroit area spending their summer in my
laboratory, through programs developed by Sam Brooks and Stuart
Ratner at the Karmanos Cancer Institute.
The project of Brandee Brewer is focused on understanding subtype differences in the function of the long terminal repeat (LTR) of the human immunodeficiency virus (HIV-1), the causative agent of AIDS. Since its origin in Africa several decades ago, the HIV-1 virus has spread out over the globe, and has evolved rapidly. At least eight phylogenetic groups ("clades") have been recognized, and classified into subtypes. The most widespread subtype is subtype C, which is present on five continents and more than 21 nations. Subtype C viruses are the most common subtype in sub-Saharan Africa, where other predominent subtypes include subtypes A and D. Subtype O and G are also present in sub-Saharan Africa to a lesser extent. Subtype C accounts for nearly all of the HIV-1 present on the Indian subcontinent, where the epidemic has moved from the former Portuguese colony of Gao to many of the major cities. More information on the global HIV-1 epidemic can be found by clicking here. The Goustin lab has recently shown that the genetic control region of the C subtype virus is markedly different from that of the B subtype virus, the subtype most prevalent in the United States and Europe:
Zacharova V, Becker MLB, Zachar V, Ebbesen P, Goustin AS. (1997). DNA sequence analysis of the long terminal repeat of the C subtype of human immunodeficiency virus type 1 from southern Africa reveals a dichotomy between B subtype and African subtypes on the basis of upstream NF-IL6 motif.
AIDS Research & Human Retroviruses 13(8), 719-724.
Brandee's project involves the construction of two infectious molecular clones of HIV-1 differing only at the -170 region of the long terminal repeat. These clones are pieces of DNA about 10,000 bp in length, which when introduced into mammalian cells direct the synthesis of replication-competent HIV-1 virus bearing the sequence of those clones as their RNA genome. Brandee's clones will be based on the strain YU-2 (available from the NIH AIDS Reference and Reagent Repository, reagent 1350, representing a patient isolate cloned from the brain of a patient with AIDS dementia (Yu et al., J. Virol. 66, 6587 (1992); accession M93258). The B subtype YU-2 virus is dual-tropic, which means that it can replicate in two of the major cell types characteristic of HIV-1 infection--T lymphocytes and monocytic cells. Monocytic cells include both blood monocytes, and their tissue derivatives known as macrophages. We hope that the YU-2 virus will also be able to replicate in dendritic cells, a key cell in the mucosal transmission pathway which we think is fundamental to transmission of the virus by the vertical (mother-to-infant) and heterosexual route.
Brandee's approach
is to divide the HIV-1 provirus into two pieces, a
large piece containing 8,400 base pairs (8.4 kb) derived from the YU-2
virus, including the genetic regions encoding the dual-tropic gp120
envelope of YU-2, and a smaller piece containing 1.6 kb derived from the
the B subtype virus HIV-1 LAI, provided as the HXB2 segment through a
collaboration with Linqui Zhang and David Ho (Huang et al., J. Virol.
69(12), 8142 (1995)) at the Aaron Diamond AIDS
Research Center in New York. Fusion of the 8.4 and 1.6 segments via
their
unique Bam HI site will permit the restoration of a full-length HIV-1
proviral clone based mostly on YU-2 and thus dual-tropic.
Brandee's immediate project is to use the Altered Sites Mutagenesis System of Promega to provide a single-stranded DNA target for oligonucleotide-mediated mutagenesis. She has prepared a 35-mer oligo (B2COLIGO) which will target the -170 region of the LTR of HXB2, including the ATTTCATCA motif, and change seven base pairs in this region to a motif similar to that seen in C subtype viruses. In this way, she hopes to eliminate the ability of C/EBP-§-related proteins such as NF-IL6 from binding to the -170 region, and thus hopes to alter the tropism of the virus, or perhaps the tempo of HIV-1 replication in T lymphocytes, monocytic cells, or dendritic cells.
Analysis of PDGF receptor dimerization in living cells using fluorescence
resonance energy transfer (FRET) of green and blue fluorescent
proteins
This is Garrett
Heffner's project. The focus of this project is to develop an
assay in living cells for the dimerization of the human ß-type
receptors for platelet-derived growth factor (PDGF). Binding of the
ligand (PDGF-AB or PDGF-BB) by this receptor brings about a rapid (few
min) re-shuffling of receptors in the plane of the lipid bilayer of
the plasma membrane, resulting in dimerization of receptors. Within
20 min, dimerization leads to activation of the intrinsic tyrosine
kinase (TK) activity of the receptors, through an autophosphorylation
cascade which results in phosphorylation of critical tyrosine (TYR)
residues in the receptor endodomain. These TYR residues include
TYR-857 within the second TK domain, and three TYR residues in the
kinase insert (KI) which separates the two halves of the TK domain.
The TYR residues in the KI domain serve as a docking site for two
downstream cellular signaling components, including the p85 regulatory
subunit of phosphatidylinositol-3-kinase (PI3K) and Ras GTPase
accelerating protein (GAP); see Figure at left. 
The mechanism of docking
of these
effectors to the KI involves the sarc homology 2 (SH2) domains in the
p85 subunit and in GAP, which form binding pockets for
phosphotyrosine. The p85 subunit docks specifically to TYR-740 and
TYR-751; GAP docks specifically to TYR-771.
This project is being continued by Poongyeon Lee, a
first year CMMG graduate student in my lab (F97).
One additional critical
signalling effector in the PDGF system is phospholipase C-
1, or PLC-1, which docks near the
C-terminus of the PDGF 
-receptor, by engaging
TYR-1009 and TYR-1021 through its own SH2 domain. Thus, PDGF receptor
activation by its ligand can be viewed as the initiation of decoration
of a receptor "Christmas tree" by multiple ornaments -receptor, bringing
together modified gfp proteins in such proximity as to allow for
fluoresence energy resonance transfer (FRET) between
spectrophotometrically-different gfp forms. FRET effects using such
gfp forms was first pioneered by Roger Tsien at the University of
California, San Diego:
Heim R, Tsien RY. (1996). Engineering green fluorescent protein for
improved brightness, longer
wavelenths and fluorescence resonance energy transfer.
In Fig. 3 of the Heim and Tsien paper (below left), they follow the loss
of the
FRET
effect by the loss of a strong green fluorescence at 507 nm as they break
the tether holding together two different forms of gfp (below, right), an
S65C mutant and
Tsien's blue fluorescent P4-3 (also known as Y66H/Y145F) mutant:
Current Biology 6:178-182
Mutants of gfp with a humanized codon bias are described by Rizzuto
et al., Current Biology 1996, Vol. 6, No 2: 183-188 (1996). Very
recently (6 AUG 97), Beat Ludin, a Swiss gfp researcher expressed his
enthusiasm for so-called 'micro-FRET' in a posting to the
fluoropro newsgroup, hinting that he knew of a paper to come out very
shortly in Nature on this subject! Also, you may wish to look at the
meeting
summary of the October 1997 gfp conference in New Jersy.
A really
cool anigif of gfp-tagged proteins by Mike Klymkowsky (University of
Colorado).
To further the mission of the Center for Molecular Medicine and Genetics
(below), I have taken a 4th Year medical student into my lab...
To foster excellence in
molecular biology,
molecular medicine, and
molecular genetics towards understanding the diagnosis,
treatment, and prevention of human diseases.
John Kalabat, M.S., Fourth Year
Medical Student
John's project is focused on a region on the short arm of human chromosome
3, in which at least five chemokine receptor genes are located, including
the gene CMKBR5
or CCR5, which encodes a seven-transmembrane (7tm)
G-protein-coupled receptor for the ß-chemokines known as RANTES, MIP-1
and MIP-1
. This gene often occurs
in the form of a non-functional allele, due to a 32-bp deletion. The
frequency of the CCR5delta32 allele varies globally, most frequently in
European populations, especially Icelandics and Ashkenazi Jews.
Amazingly, delta32/delta32 homozygotes are not immunologically compromised
at all, a fact explained by many researchers by reference to unknown gene
redundancies, for example other chemokines receptors, which take over for
CCR5 when it is knocked out.
The Goustin laboratory is located on the Fifth Floor of the Biological
Sciences Building, WSU Main Campus, across from the newly-renovated
historic Old Main (below).
For
more information about positions available, contact:
Anton
Scott Goustin, Ph.D. (313-993-7688)
asg@cmb.biosci.wayne.edu
