1. Variable positions in U1A6 snRNA Qie Kuang Qie Kuang 12-17-2005 12-17-2005

2. Lodish et al. Molecular Cell Biology, 5th ed

3. Introduction In early experiment, Kyriakopoulou and his co- workers had cloned an U1-like snRNA sequence: U1A6, which has 72% similarities to the gene coded by the HUMUR1A (U1A). Considering they only tested once, so we repeated the experiment, hoping to find some new variable positions of U1A6 and their correlation to U1A6’ structure. In early experiment, Kyriakopoulou and his co- workers had cloned an U1-like snRNA sequence: U1A6, which has 72% similarities to the gene coded by the HUMUR1A (U1A). Considering they only tested once, so we repeated the experiment, hoping to find some new variable positions of U1A6 and their correlation to U1A6’ structure.

4. Materials and methods Samples Samples Samples Restriction Restriction Restriction Amplify by PCR Amplify by PCR Amplify by PCR Amplify by PCR Test in Agarose Gel Electrophoresis Test in Agarose Gel Electrophoresis Test in Agarose Gel Electrophoresis Test in Agarose Gel Electrophoresis Gel excision and TA cloning Gel excision and TA cloning Gel excision and TA cloning Gel excision and TA cloning Over night culturing E. coli, preparing plasmid DNA and sequence Over night culturing E. coli, preparing plasmid DNA and sequence Over night culturing E. coli, preparing plasmid DNA and sequence Over night culturing E. coli, preparing plasmid DNA and sequence

5. The following samples were added: The following samples were added: 1. human male DNA (given from Company Stratagene). 1. human male DNA (given from Company Stratagene). 2.Chimp DNA (ID:38114) (given from Wolfgang Enard, Max-Planck- institute for Evolutionary Anthropology, Leipzy, Germany) 2.Chimp DNA (ID:38114) (given from Wolfgang Enard, Max-Planck- institute for Evolutionary Anthropology, Leipzy, Germany) 3.Individual DNA (given from Lotta Thuresson Rudbeck Lab, Uppsala university) 3.Individual DNA (given from Lotta Thuresson Rudbeck Lab, Uppsala university)

6. Restriction Restriction Restriction Approximately 100 μg of genomic DNA were incubated in the presence of 1μg Pst1 as restriction enzyme. Approximately 100 μg of genomic DNA were incubated in the presence of 1μg Pst1 as restriction enzyme. Pst1 Pst1

7. Amplify by PCR Amplify by PCR Amplify by PCR Amplify by PCR Js19 Js20

8. Test in 1% Agarose Gel Electrophoresis Test in 1% Agarose Gel Electrophoresis Test in 1% Agarose Gel Electrophoresis Test in 1% Agarose Gel Electrophoresis Sample Negative control Aim band

9. Gel excisions and TA clone Gel excisions and TA clone Gel excisions and TA clone Gel excisions and TA clone Sample Negative control Gel excision TA Cloning

10. Over night culturing E.coli, preparing plasmid DNA and sequence Over night culturing E.coli, preparing plasmid DNA and sequence Over night culturing Over night culturing Extract plasmid DNA Extract plasmid DNA Sequence Sequence

11. Result and discussion A typical picture of 1% electrophoresis. A typical picture of 1% electrophoresis.

12. Human U1A6 alignment Human U1A6 alignmentHuman U1A6 alignmentHuman U1A6 alignment 1.U1A6 may have different ways of splicing 1.U1A6 may have different ways of splicing 2. 11 variable nucleotides correlates with C8 and T71 2. 11 variable nucleotides correlates with C8 and T71 Chimp and Human U1A6 alignment Chimp and Human U1A6 alignment Chimp and Human U1A6 alignment Chimp and Human U1A6 alignment The 11 variable nucleotides is conserved and probably inherited from our ancestor. The 11 variable nucleotides is conserved and probably inherited from our ancestor.

15. Sumary Our studies proved that U1A6 gene may have two different alleles, one has an 11 variable nucleotide region and the other does not have. And these 11 variable nucleotides which correlates with C8 and T71 is highly conserved in both human and chimp. Our studies proved that U1A6 gene may have two different alleles, one has an 11 variable nucleotide region and the other does not have. And these 11 variable nucleotides which correlates with C8 and T71 is highly conserved in both human and chimp. THANKS! THANKS!