Characterization of novel DNA-binding proteins expressed in snake oocyte cDNA library.
DNA-binding proteins play pivotal roles in transcription, DNA-replication, recombination/repair and determine cell-fate in all physiological conditions of differentiation, development and disease. As they are present in extremely small amounts in cells, their isolation/identification, particularly from scarce tissues is impracticable. We cloned the cDNA pool of snake (Ptyas mucosus) oocytes (a scarce tissue) in bacteria, overexpressed total library, purified and identified DNA-binding proteins expressed in the library. Although snake databases do not exist, we identified 23 DNA-binding proteins, obtained 10-15 amino acids internal sequence tags of six of them and succeeded in PCR amplification of the cDNAs of five proteins. We employed electro spray ionization mass spectrometry, matrix assisted laser desorption/ionization time of flight and analyzed the results by peptide mass fingerprint (PMF) and various sequence BLAST analyses. Proteins identified were largely unanimous between the PMF and BLAST analyses. We expect these proteins to play important roles in snake embryonic development and differentiation. We arrived at homologous mouse proteins to some of the identified snake proteins and are working towards characterizing their structure and physiological function. Similar approaches shall prove valuable in isolation and identification of important factors from scarce carcinoma tissues, mammalian oocytes and early embryos, which might be involved in important functions like nuclear reprogramming, embryonic development and differentiation.
Ganesan
Mala
M
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.
Paithankar
Khanderao R
KR
Jagannadham
Medicharla V
MV
Sundaram
Curam S
CS
Murthy
Bulusu S
BS
Singh
Lalji
L
D000818:Animals; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017812:Colubridae / Q000196:embryology / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004625:Embryo, Nonmammalian; D005260:Female; D015723:Gene Library; D009865:Oocytes / Q000378:metabolism*; D010449:Peptide Mapping; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000494:pharmacology
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D014357:Trypsin