This experiment aimed to identify genes that were overexpressed in BU.MPT cells resistant to apoptosis compared to normal BU.MPT cells. RNA from the two cell populations underwent subtractive hybridization to identify differences. Eight colonies were sequenced, with one gene of interest identified as cysteine and histidine rich protein 1 (Chrp). Chrp is known to bind specifically to galectin-3, keeping it localized in the cytoplasm where it can activate anti-apoptotic pathways and properties. This binding of Chrp to galectin-3 provides a potential explanation for the resistance to apoptosis in the selected BU.MPT cells.

1. Acknowledgments
Introduction
Apoptosis is the process of programmed cell death
that rids a multicellular organism of unhealthy cells.
Apoptosis becomes abnormal when too many or too
few cell deaths occur. Many autoimmune diseases are
believed to be related to excessive apoptosis.
Apoptosis that does not occur usually leads to tumor
formation and cancer.
Two BU.MPT cell populations were used in this
experiment: normal BU.MPT cells that readily
underwent apoptosis, and selected BU.MPT cells
which resisted apoptosis.
The goal of our experiment was to clone and
sequence genes that were overexpressed in the two
cell populations. RNA sequences that were the same
were removed from the two populations.
Anything that was different from the other
population was cloned. A method called subtractive
hybridization was used to amplify PCR-based cDNA
fragments from two closely related samples
Project Objectives
• To clone any overexpressed genes that were present
after subtractive hybridization took place
Methods
RNA Extraction and Purification
• The RNA was extracted and purified by a
method that used the Trizol Reagent from
Invitrogen
Synthesis of cDNA and DNA amplification
• A Super SMART cDNA synthesis kit from
Clontech was used to synthesize the first strand
of cDNA
Restriction Digest with Rsa1
• The restriction digested occurred with RsaI as
per the Clontech Protocol
Ligation of Linkers to Tester cDNA
• Linkers were added to the ends of the cDNA by
using the Clontech Subtractive Hybridization kit
• PCR amplification was used to analyze the
efficiency of ligating the adaptors
• Gel electrophoresis was performed to analyze the
DNA after the second PCR amplification
Subtractive Hybridization
• Subtractive hybridization was used to compare
the two cDNA populations (tester and driver)
Clone Sequencing & Analysis
• Isolated colonies were transferred onto new LB +
Kanamycin plates and grown overnight.
• Colonies that grew well were transferred to a new
LB + Kanamycin broth culture and were grown
overnight.
• The clones were sent out to Sequetech for
sequencing using the T3 primer.
Discussion
Of the 8 tester clones that were sequenced and
analyzed, Apo 18 gave the most interesting result:
• Cystidine and histidine rich protein (Chrp)
• Is found distributed throughout the cytoplasm &
concentrated in a ring at the nuclear envelope
Chrp is known to interact directly with another protein
called galectin-3
• Galectin-3 is usually be found in the cytoplasm
• Shown interact with signaling pathways and to
regulate cell cycle and growth
Galectin-3 has shown to have anti-apoptotic as well as
pro-apoptotic properties depending on where it is
expressed in the cell
• Anti-apoptotic if expressed intracellularly
• Pro-apoptotic if expressed extracellularly
Chrp binds exclusively to the carbohydrate recognition
domain (CRD) of galectin-3
• Chrp does not bind to other galectins with similar
CRD sequences
• Suggests a strong relationship
The Search of Overexpressed Genes in BU.MPT Cells Using Selective Hybridization:The
Significance of Chrp in Relation to Apoptosis
Marcus Bowie andTaren Fritzinger
Department of Biology, Kutztown University, Kutztown PA, 19530
Literature cited
• Sulemana Bawumia, Eminia A.M. Barboni , Rajesh P. Menon, R.
Colin Hughes “Specificity of interactions of galectin-3 with Chrp,
a cysteine- and histidine-rich cytoplasmic protein” National
Institute for Medical Research, 2003, pdf.web.
• Fu-Tong Liu a, Ronald J. Patterson , John L. Wang “Intracellular
functions of galectins” aDepartment of Dermatology, University of
California, Davis, School of Medicine, Department of
Microbiology and Molecular Genetics, Michigan State University,
Department of Biochemistry, Michigan State University, 2002,
pdf.web.
• Amy Dhirapong , Ana Lleo , Patrick Leung , M. Eric Gershwin ,
Fu-Tong Liu “The immunological potential of galectin-1 and -3”
Division of Rheumatology, Allergy and Clinical Immunology,
University of California at Davis, School of Medicine,
Department of Dermatology, University of California at Davis,
School of Medicine, 2008, pdf.web.
Results
Sequenced Genes
• Out of the 32 colonies cultured, 8 that had healthy
growth were collected and sent out to be
sequenced
• Genes were isolated and identified by using a
nucleotide BLAST search
Clone Gene
Apo 17 Mus musculus ribosomal protein S2 (Rps2), mRNA
*Apo 18 Mus musculus cysteine and histidine rich 1 (Cyhr 1),
transcript variant 3, mRNA
Apo 21 Mus musculus polymerase (DNA directed)m gamma 2,
accessory subunit (Polg 2), transcript variant 1, mRNA
Apo 23 Mus musculus class Ib MHC antigen Qa-2 (Q6) gene,
complete cds
Apo 26 Mus musculus targeted KO-first, conditional ready, lacZ-
tagged mutant allele Unc13d:tml1a(KOMP)Wtsi;
transgenic
Apo 27 Mus musculus cell-line Nuero-2a 18S ribosomal RNA,
complete sequence
Apo 28 Mus musculus 45S pre-ribosomal RNA (Rn45s),
ribosomal RNA
Apo 29 Mus musculus eukaryotic translation elongation factor 1
beta 2 (Eef1b2), mRNA
Figure 2:Nucleotide sequence of the Chrp gene that was obtained from the
NCBI BLAST search
Table 1: The table below shows the genes that have been isolated and sequenced
from 8 bacterial colonies on LB + Kanamycin plates. Apo 18 showed a particular
interest.
We would like to thank Dr. Antoni, the
Department of Biology, Kutztown University, and
Dr. Jerrold Levine from the University of Chicago
for helping to make this research possible.
Conclusion
Figure 1: Results of gel electrophoresis with lanes 2-8 containing normal BU-
MPT, lanes 9-15 containing selected BU-MPT, and lanes 1 & 16 containing the
Hi-Lo DNA ladders.
• The Chrp gene was found to be
overexpressed in selected BU-MPT
• Chrp shown to uniquely bind to
galectin-3
• Keeps galectin-3 bound in the
cytoplasm to activate and utilize its
anti-apoptotic properties
• Explains how selected BU-MPT can
block apoptosis internally