Acknowledgments

IntroductionApoptosis 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

MethodsRNA 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 ProtocolLigation 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 amplificationSubtractive 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.

DiscussionOf 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 envelopeChrp 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 extracellularlyChrp 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 and Taren FritzingerDepartment 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.

ResultsSequenced 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 GeneApo 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