INGAP protein involved in pancreatic islet neogenesis

Abstract

Cellophane wrapping (CW) of hamster pancreas induces proliferation of duct epithelial cells followed by endocrine cell differentiation and islet neogenesis. Using the mRNA differential display technique a cDNA clone expressed in cellophane wrapped but not in control pancreata was identified. Using this cDNA as a probe, a cDNA library was screened and a gene not previously described was identified and named INGAP.

1. An isolated DNA molecule encoding a mammalian islet cell neogenesis associated protein (INGAP) protein , wherein the  INGAP protein  has the amino acid sequence shown in SEQ ID NO: 2. 
     
     
       2. The DNA molecule of  claim 1  wherein the INGAP protein  which has the nucleotide sequence shown in SEQ ID NO: 1. 
     
     
       3. A vector comprising the DNA of  claim 1 . 
     
     
       4. The vector of  claim 3  further comprising expression control sequences, whereby said DNA is expressed in a host cell. 
     
     
       5. The vector of  claim 4  which comprises a  an Epstein Barr Nuclear Antigen- Histidine  (EBNA His) plasmid. 
     
     
       6. A  An isolated host cell transformed with the DNA of  claim 1 . 
     
     
       7. A  An isolated host cell transformed with the vector of  claim 3 . 
     
     
       8. The host cell of  claim 6  which is a cos7,African  cos 7 , African Green Monkey kidney cell. 
     
     
       9. A nucleotide probe comprising at least 30 contiguous nucleotides of a sequence encoding a mammalian islet cell neogenesis associated protein (INGAP), wherein said protein has the sequence shown in SEQ ID NO: 2. 
     
     
       10. The nucleotide probe of  claim 9  wherein the nucleotide sequence encoding a mammalian INGAP gene  has the sequence shown in SEQ ID NO: 1. 
     
     
       11. The nucleotide probe of  claim 9  wherein said probe is labeled with a detectable moiety. 
     
     
       12. A  An isolated DNA molecule comprising at least 30 contiguous nucleotides of a sequence encoding a mammalian islet cell neogenesis associated protein (INGAP), wherein said protein has the sequence shown in SEQ ID NO:  2, wherein said DNA molecule encodes a polypeptide which stimulates islet cell neogenesis.    
     
     
       13. The DNA molecule of  claim 12  wherein the sequence encoding the mammalian INGAP gene  has the sequence shown in SEQ ID NO: 1. 
     
     
       14. The DNA molecule of  claim 12  wherein said molecule is labeled with a detectable moiety. 
     
     
       15. A method of producing a mammalian INGAP protein , comprising the steps of:
 providing a host cell according to  claim 6 ;  
 culturing the host cell in a nutrient medium so that the INGAP protein  is expressed; and  
 harvesting the INGAP protein  from the host cells or the nutrient medium.  
 
     
     
       16. A method of producing a mammalian INGAP protein , comprising the steps of:
 providing a host cell comprising the DNA molecule of  claim 1 ;  
 culturing the host cell in a nutrient medium so that the mammalian INGAP protein  is expressed; and  
 harvesting the mammalian INGAP protein  from the host cells or the nutrient medium.  
 
     
     
       17. An antisense construct of a mammalian islet cell neogenesis associated protein (INGAP) gene comprising:
 a promoter, a terminator, and a nucleotide sequence consisting of a mammalian INGAP gene, wherein the gene  which encodes all or a portion of a protein as shown in SEQ ID NO: 2, said nucleotide sequence being between said promoter and said terminator, said nucleotide sequence being inverted with respect to said promoter, whereby upon expression from said promoter an mRNA complementary to native mammalian INGAP mRNA is produced, wherein said mRNA complementary to native mammalian INGAP mRNA prevents translation of the native mammalian INGAP mRNA.  
 
     
     
       18. The DNA molecule of  claim 1  wherein the INGAP protein  is from human. 
     
     
       19. The DNA molecule of  claim 1  which comprises nucleotides 4 to 268 and 389 to 629 of SEQ ID NO: 1. 
     
     
       20. A vector comprising the DNA of  claim 2 . 
     
     
       21. An isolated host cell transformed with the vector of  claim 20 . 
     
     
       22. The DNA molecule of  claim 1  which is a cDNA molecule. 
     
     
       23. The DNA molecule of  claim 12  which is a cDNA molecule. 
     
     
       24. The DNA molecule of  claim 12  which encodes a portion of INGAP, wherein said DNA molecule encodes a polypeptide which stimulates islet cell neogenesis.