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Molecular Biology of Gut Peptides

Randy S. Haun, Carolyn D. Minth, Philip C. Andrews, Jack E. Dixon

10.1002/cphy.cp060201

Source: Supplement 17: Handbook of Physiology, The Gastrointestinal System, Neural and Endocrine Biology

Originally published: 1989

Published online: January 2011

Full Article on Wiley Online Library



Abstract

The sections in this article are:

1 Methods in Molecular Biology
1.1 Construction of a cDNA Library
1.2 Identifying Specific cDNA Clones
1.3 Construction of Genomic Libraries
1.4 Isolation of Cloned Genes
1.5 Structure of Eucaryotic Genes
2 Gene Expression
3 Gastrin/Cholecystokinin Family
3.1 Molecular Biology of Gastrin
3.2 Molecular Biology of Cholecystokinin
4 Pancreatic Polypeptide, Neuropeptide Y, and Peptide YY Family
5 Glucagon/Secretin Family
5.1 Glucagon
5.2 Growth Hormone‐Releasing Factor
6 Summary
Figure 1. Figure 1.

Sequencing end‐labeled DNA by limited, base‐specific cleavage. A: three consecutive reactions cleave one DNA molecule at one guanine. B: nested set of end‐labeled fragments generated when these reactions cleave at a guanine in all such molecules.

From Zubay 209a. In: Biochemistry. © 1983, reprinted by permission of Addison‐Wesley Publishing Company, Inc., Reading, MA, Fig. 20,21,22,23,24,25,26,27,28,29,30,31,32 33, 34
Figure 2. Figure 2.

Sanger DNA‐sequencing procedure. A: preparation of series of labeled fragments having identical starting points but of variable length. B: DNA strand to be sequenced, along with labeled primer, is split into four DNA polymerase reactions, each containing one of the four ddNTPs. Resultant labeled fragments are separated by size on acrylamide gel, autoradiography is performed, and pattern of fragments gives DNA sequence.

From Recombinant DNA: A Short Course, by James D. Watson et al. 197a. Copyright © 1983, James D. Watson, John Tooze, and David T. Kurtz. Reprinted with the permission of Scientific American Books, New York
Figure 3. Figure 3.

Diagram of the E. coli plasmid cloning vehicle pUC19. Molecule is double‐stranded DNA circle 2,686 base pairs in length. Nucleotide numbering starts at first T in sequence TCGCGCGTTT and proceeds clockwise around molecule in direction lac to Ap. Map shows restriction sites of those enzymes that cut molecule once or twice; unique sites are in bold type. Polylinker is shown below map. Map also shows position of coding sequences and origin of replication.

From New England Biolabs Catalog 1986/1987, Beverly, MA, p. 94
Figure 4. Figure 4.

Synthesis of double‐stranded cDNA from mRNA. Oligo‐dT segment serves as primer for action of reverse transcriptase, which uses mRNA as a template for synthesis of complementary DNA strand. When mRNA strand is degraded by treatment with NaOH, hairpin loop becomes primer for DNA polymerase I, which completes paired DNA strand. Loop is then cleaved by S1 nuclease to produce double‐stranded cDNA molecule.

From Recombinant DNA: A Short Course, by James D. Watson et al. 197a. Copyright © 1983, James D. Watson, John Tooze, and David T. Kurtz. Reprinted with the permission of Scientific American Books, New York
Figure 5. Figure 5.

Preparation of cDNA clone into DNA plasmid by homopolymer tailing.

From Molecular Cell Biology, by James E. Darnell et al. 31a. Copyright © 1986. Scientific American Books. Used by permission
Figure 6. Figure 6.

Preparation of probe to isolate rare cDNA. mRNA for protein α2u‐globulin represents 1% of mRNA in adult male rat liver but is absent from female rat liver. Male rat liver mRNA is reverse‐transcribed into cDNA with labeled nucleotides; cDNA is then hybridized with excess of female liver mRNA. Most mRNA sequences in male and female liver are common, so most of male cDNA hybridizes with mRNA from female liver. However, α2u cDNA remains single stranded and can be separated from cDNA‐mRNA hybrids by chromatography on hydroxyapatite, which binds double‐stranded molecules much more avidly than single‐stranded molecules. Labeled α2u cDNA can then be used to screen male rat liver cDNA library to obtain cloned copy of α2u cDNA.

From Recombinant DNA: A Short Course, by James D. Watson et al. 197a. Copyright © 1983, James D. Watson, John Tooze, and David T. Kurtz. Reprinted with the permission of Scientific American Books, New York
Figure 7. Figure 7.

Immunochemical screening of cDNA library cloned in a bacterial expression vector.

From Recombinant DNA: A Short Course, by James D. Watson et al. 197a. Copyright © 1983, James D. Watson, John Tooze, and David T. Kurtz. Reprinted with the permission of Scientific American Books, New York
Figure 8. Figure 8.

Generalized structure of eucaryotic expression vector pcD‐cDNA recombinants. Portion of hatched region containing SV40 origin of DNA replication (ori) and early and late promoters corresponds to SV40 segment between PvuII and HindIII restriction sites at map positions 0.71 to 0.65; other portion of hatched region joining first on early region promoter side derives from SV40 late region between map positions 0.75 and 0.95, with BamHI sequence replacing internal region between map positions 0.77 and 0.93. For use as linker segment, hatched fragment contains HindIII cohesive end and oligo(dG) sequence at PstI terminus. Solid dark region, cloned cDNA; stippled region, segment carrying SV40 late region polyadenylation signal.

From Okayama and Berg 145
Figure 9. Figure 9.

Identification of bacterial colonies harboring specific cDNA plasmid by hybridization with 32P‐labeled cDNA, mRNA, or oligonucleotide probe.

From Recombinant DNA: A Short Course, by James D. Watson et al. 197a. Copyright © 1983, James D. Watson, John Tooze, and David T. Kurtz. Reprinted with the permission of Scientific American Books, New York
Figure 10. Figure 10.

Identification of clones harboring somatostatin cDNA. A: electrophoresis of cDNA synthesized from rat thyroid medullary carcinoma poly(A) RNA primed with 5'‐32P‐labeled oligonucleotide, d(T‐T‐C‐C‐A‐G‐A‐A‐G‐A‐A). Arrow denotes primer‐extended cDNA used for colony hybridization. B: autoradiograph of in situ colony hybridization of bacterial transformants. Radiolabeled primer‐extended cDNA was used as hybridization probe.

From Funckes et al. 53
Figure 11. Figure 11.

Composite restriction map of pRT B1‐63, pRT 3–36, and pRT 3–81. Sequencing strategy is shown below restriction map. Boxed area represents preprohormone; dark area denotes area corresponding to somatostatin‐14.

From Funckes et al. 53
Figure 12. Figure 12.

Strategy for constructing libraries of random fragments of eukaryotic DNA. Left, preparation of vector DNA fragments; right, preparation of eukaryotic DNA fragments. Concatameric, recombinant DNA molecule is produced by action of bacteriophage T4 DNA ligase. This concatamer is substrate for in vitro packaging reaction during which a different recombinant DNA molecule is inserted into each bacteriophage particle. After amplification by growth in E. coli, lysate is obtained consisting of library of recombinant clones that in aggregate contain most sequences present in mammalian genome.

From Maniatis et al. 111a
Figure 13. Figure 13.

Map of rat somatostatin gene. Restriction sites in Λb clone were determined by combination of Southern hybridization of digested DNA and partial digestion of end‐labeled fragments. 5'‐to‐3’ Orientation of somatostatin gene within clone is indicated. Two exons of gene are indicated by solid boxes.

From Tavianini et al. 186
Figure 14. Figure 14.

Determination of transcription initiation site by nuclease protection assay.

From Lewin 102a, reprinted by permission of John Wiley & Sons, Inc. © 1986
Figure 15. Figure 15.

Introns spliced out during maturation of mRNA.

From Recombinant DNA: A Short Course, by James D. Watson et al. (197a.) Copyright © 1983, James D. Watson, John Tooze, and David T. Kurtz. Reprinted with the permission of Scientific American Books, New York
Figure 16. Figure 16.

Construction of recombinant pBXΔ‐750. Plasmid pBXΔ‐750 was constructed from pRSΛHE5, a Λ phage subclone, containing 750‐base‐pair fragment (bp) of 5’ upstream sequences and first exon of rat somatostatin gene. Unique XbaI site at +50 in pRSΛHE5 was converted to BamHI site and 800 bp HindIII‐BamHI fragment was cloned into unique BamHI, HindIII sites of promoterless pBR322 derivative CAT plasmid, resulting in plasmid pBXΔ‐750.
Figure 17. Figure 17.

Promoter for RNA polymerase II containing separate sequence components with different roles. Sequences between components are not important. Orientation of conserved sequences is indicated by direction of arrows. Transcription factors bind to the conserved sequence motifs.

From Lewin 102a, reprinted by permission of John Wiley & Sons, Inc. © 1986
Figure 18. Figure 18.

Identification of DNA binding sites by DNase I footprinting.

From Lewin 102a, reprinted by permission of John Wiley & Sons, Inc. © 1986
Figure 19. Figure 19.

Splice commitment regulatory factor model for cell‐specific alternative RNA processing of a complex transcription unit in the brain. Putative factor that commits exon splicing pattern is represented by stippled oval and is postulated to inhibit usage of calcitonin poly(A) site.

From Leff et al. 99). Copyright 1987, by permission of Cell Press
Figure 20. Figure 20.

Nucleotide and amino acid sequence alignments of rat tumor cholecystokinin (CCK) and porcine gut gastrin (GSN) preprohormones. Amino acid identities are boxed. The putative initiator methionine in each case is designated +1. Deletions are indicated by dashed line.

From Deschenes et al. 34). Reprinted by permission of Elsevier Science Publishing Co., copyright 1985
Figure 21. Figure 21.

Schematic representation showing exon organization of human gastrin and rat cholecystokinin (CCK) genes. Second exons are aligned at putative initiator methionines, and third exons are aligned at conserved pentapeptide of preprohormones. Shaded boxes indicate translated exons, open boxes indicate nontranslated exons, and cross‐hatched boxes indicate conserved amino acids in preprohormones.
Figure 22. Figure 22.

Comparison of porcine amino acid sequence of neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PPP). Identities are underlined.
Figure 23. Figure 23.

Nucleotide sequence of pancreatic polypeptide cDNA clone. Restriction endonuclease sites for BamHI, HinfI, and AvaII, which were used for DNA sequencing, and oligonucleotide sequence corresponding to probe used for screening are indicated by solid lines. Deduced amino acid sequence is numbered from NH2‐terminal alanine of pancreatic polypeptide portion of sequence. Dashed lines, proposed leader sequence and icosapeptide portion; boxes, connecting sequence (Gly‐Lys‐Arg) and variant amino acid.

From Takeuchi and Yamada 182).
Figure 24. Figure 24.

Restriction map of human pancreatic polypeptide gene. Restriction sites in pPP4A subclone are shown. Four exons indicated as cross‐hatched boxes; repetitive DNA (Rep DNA) sequences shown as solid box and labeled; DNA sequencing strategy indicated by arrows below map; Solid arrows indicate 5'‐labeled fragments; and dashed arrows denote fragments labeled at 3’ ends base pairs (bp)

From Leiter et al. 101
Figure 25. Figure 25.

Nucleotide sequence encoding precursor to human NPY. Amino acid sequence of preproNPY numbered 1 through 97; termination codon denoted by asterisks; mature hormone is underlined; putative signal for poly(A) addition is underlined with broken line.

From Minth et al. 125
Figure 26. Figure 26.

Diagram of splicing of human neuropeptide Y (NPY) gene. Exons are represented as boxes, and dotted lines indicate how exons are spliced to produce mRNA; 5’ nontranslated sequences are located on exon 1 and shown as open box; second exon codes for signal peptide, residues 1–28, stippled box; mature NPY, residues 29–63, cross‐hatched box; third exon contains coding region for amino acid residues 64–90, solid box; fourth exon contains COOH‐terminal heptapeptide, residues 91–97, solid box; and 3’ nontranslated region, open box, including poly(A) addition site.

From Minth et al. 124
Figure 27. Figure 27.

Diagram of amino acid sequences and exon‐intron junctions of human neuropeptide Y (NPY) and human pancreatic polypeptide (PP). Boxes, identical amino acids. Splicing of gene after first two nucleotides of Arg codons depicted by splitting the three‐letter code for Arg after the first two letters (Ar‐g). Introns are represented by lines; their respective sizes are noted.

From Minth et al. 124
Figure 28. Figure 28.

Structures of two anglerfish preproglucagons 21,22 deduced from their respective cDNAs (AFG I and AFG II). Boxes, regions of protein sequence identity; arrows, prohormone processing sites.

From Lund et al. 109
Figure 29. Figure 29.

Structure of rat preproglucagon deduced from cDNA to rat preproglucagon. Circles, putative prohormone conversion sites. Identities of peptides indicated are NH2‐peptide, glicentin‐related polypeptide; IP‐I, intervening peptide‐I; IP‐II, intervening peptide‐II; GLP‐I, glucagon‐like peptide‐I; GLP‐II, glucagon‐like peptide‐II

From Heinrich et al. 72). Copyright 1984 by permission of The Endocrine Society
Figure 30. Figure 30.

Partial sequence of rat preproglucagon gene. Locations of exons indicated by capitalized bases and three‐letter code for deduced amino acids. Introns indicated by lowercase bases. Signal cleavage site (between −1 and +1) indicated by arrow, as are putative prohormone processing sites. Diagram at bottom summarizes exon‐intron structure of rat preproglucagon gene. Known or suspected bioactive peptides, solid boxes; nonhormone peptide portions, hatched boxes; signal peptide, stippled boxes; and noncoding portions of mRNA, open boxes.

From Heinrich et al. 71
Figure 31. Figure 31.

Structures of two related clones of human preproGRF. Bioactive regions enclosed by a light‐lined box; putative prohormone processing sites enclosed by dark‐lined boxes; differences between clones indicated by arrows; glycine that donates COOH‐terminal amide nitrogen indicated by asterisk; putative signal peptides underlined.

From Gubler et al. 66
Figure 32. Figure 32.

Sequence of human gene for preproGRF. Exons indicated by large characters.

From Mayo et al. 114


Figure 1.

Sequencing end‐labeled DNA by limited, base‐specific cleavage. A: three consecutive reactions cleave one DNA molecule at one guanine. B: nested set of end‐labeled fragments generated when these reactions cleave at a guanine in all such molecules.

From Zubay 209a. In: Biochemistry. © 1983, reprinted by permission of Addison‐Wesley Publishing Company, Inc., Reading, MA, Fig. 20,21,22,23,24,25,26,27,28,29,30,31,32 33, 34


Figure 2.

Sanger DNA‐sequencing procedure. A: preparation of series of labeled fragments having identical starting points but of variable length. B: DNA strand to be sequenced, along with labeled primer, is split into four DNA polymerase reactions, each containing one of the four ddNTPs. Resultant labeled fragments are separated by size on acrylamide gel, autoradiography is performed, and pattern of fragments gives DNA sequence.

From Recombinant DNA: A Short Course, by James D. Watson et al. 197a. Copyright © 1983, James D. Watson, John Tooze, and David T. Kurtz. Reprinted with the permission of Scientific American Books, New York


Figure 3.

Diagram of the E. coli plasmid cloning vehicle pUC19. Molecule is double‐stranded DNA circle 2,686 base pairs in length. Nucleotide numbering starts at first T in sequence TCGCGCGTTT and proceeds clockwise around molecule in direction lac to Ap. Map shows restriction sites of those enzymes that cut molecule once or twice; unique sites are in bold type. Polylinker is shown below map. Map also shows position of coding sequences and origin of replication.

From New England Biolabs Catalog 1986/1987, Beverly, MA, p. 94


Figure 4.

Synthesis of double‐stranded cDNA from mRNA. Oligo‐dT segment serves as primer for action of reverse transcriptase, which uses mRNA as a template for synthesis of complementary DNA strand. When mRNA strand is degraded by treatment with NaOH, hairpin loop becomes primer for DNA polymerase I, which completes paired DNA strand. Loop is then cleaved by S1 nuclease to produce double‐stranded cDNA molecule.

From Recombinant DNA: A Short Course, by James D. Watson et al. 197a. Copyright © 1983, James D. Watson, John Tooze, and David T. Kurtz. Reprinted with the permission of Scientific American Books, New York


Figure 5.

Preparation of cDNA clone into DNA plasmid by homopolymer tailing.

From Molecular Cell Biology, by James E. Darnell et al. 31a. Copyright © 1986. Scientific American Books. Used by permission


Figure 6.

Preparation of probe to isolate rare cDNA. mRNA for protein α2u‐globulin represents 1% of mRNA in adult male rat liver but is absent from female rat liver. Male rat liver mRNA is reverse‐transcribed into cDNA with labeled nucleotides; cDNA is then hybridized with excess of female liver mRNA. Most mRNA sequences in male and female liver are common, so most of male cDNA hybridizes with mRNA from female liver. However, α2u cDNA remains single stranded and can be separated from cDNA‐mRNA hybrids by chromatography on hydroxyapatite, which binds double‐stranded molecules much more avidly than single‐stranded molecules. Labeled α2u cDNA can then be used to screen male rat liver cDNA library to obtain cloned copy of α2u cDNA.

From Recombinant DNA: A Short Course, by James D. Watson et al. 197a. Copyright © 1983, James D. Watson, John Tooze, and David T. Kurtz. Reprinted with the permission of Scientific American Books, New York


Figure 7.

Immunochemical screening of cDNA library cloned in a bacterial expression vector.

From Recombinant DNA: A Short Course, by James D. Watson et al. 197a. Copyright © 1983, James D. Watson, John Tooze, and David T. Kurtz. Reprinted with the permission of Scientific American Books, New York


Figure 8.

Generalized structure of eucaryotic expression vector pcD‐cDNA recombinants. Portion of hatched region containing SV40 origin of DNA replication (ori) and early and late promoters corresponds to SV40 segment between PvuII and HindIII restriction sites at map positions 0.71 to 0.65; other portion of hatched region joining first on early region promoter side derives from SV40 late region between map positions 0.75 and 0.95, with BamHI sequence replacing internal region between map positions 0.77 and 0.93. For use as linker segment, hatched fragment contains HindIII cohesive end and oligo(dG) sequence at PstI terminus. Solid dark region, cloned cDNA; stippled region, segment carrying SV40 late region polyadenylation signal.

From Okayama and Berg 145


Figure 9.

Identification of bacterial colonies harboring specific cDNA plasmid by hybridization with 32P‐labeled cDNA, mRNA, or oligonucleotide probe.

From Recombinant DNA: A Short Course, by James D. Watson et al. 197a. Copyright © 1983, James D. Watson, John Tooze, and David T. Kurtz. Reprinted with the permission of Scientific American Books, New York


Figure 10.

Identification of clones harboring somatostatin cDNA. A: electrophoresis of cDNA synthesized from rat thyroid medullary carcinoma poly(A) RNA primed with 5'‐32P‐labeled oligonucleotide, d(T‐T‐C‐C‐A‐G‐A‐A‐G‐A‐A). Arrow denotes primer‐extended cDNA used for colony hybridization. B: autoradiograph of in situ colony hybridization of bacterial transformants. Radiolabeled primer‐extended cDNA was used as hybridization probe.

From Funckes et al. 53


Figure 11.

Composite restriction map of pRT B1‐63, pRT 3–36, and pRT 3–81. Sequencing strategy is shown below restriction map. Boxed area represents preprohormone; dark area denotes area corresponding to somatostatin‐14.

From Funckes et al. 53


Figure 12.

Strategy for constructing libraries of random fragments of eukaryotic DNA. Left, preparation of vector DNA fragments; right, preparation of eukaryotic DNA fragments. Concatameric, recombinant DNA molecule is produced by action of bacteriophage T4 DNA ligase. This concatamer is substrate for in vitro packaging reaction during which a different recombinant DNA molecule is inserted into each bacteriophage particle. After amplification by growth in E. coli, lysate is obtained consisting of library of recombinant clones that in aggregate contain most sequences present in mammalian genome.

From Maniatis et al. 111a


Figure 13.

Map of rat somatostatin gene. Restriction sites in Λb clone were determined by combination of Southern hybridization of digested DNA and partial digestion of end‐labeled fragments. 5'‐to‐3’ Orientation of somatostatin gene within clone is indicated. Two exons of gene are indicated by solid boxes.

From Tavianini et al. 186


Figure 14.

Determination of transcription initiation site by nuclease protection assay.

From Lewin 102a, reprinted by permission of John Wiley & Sons, Inc. © 1986


Figure 15.

Introns spliced out during maturation of mRNA.

From Recombinant DNA: A Short Course, by James D. Watson et al. (197a.) Copyright © 1983, James D. Watson, John Tooze, and David T. Kurtz. Reprinted with the permission of Scientific American Books, New York


Figure 16.

Construction of recombinant pBXΔ‐750. Plasmid pBXΔ‐750 was constructed from pRSΛHE5, a Λ phage subclone, containing 750‐base‐pair fragment (bp) of 5’ upstream sequences and first exon of rat somatostatin gene. Unique XbaI site at +50 in pRSΛHE5 was converted to BamHI site and 800 bp HindIII‐BamHI fragment was cloned into unique BamHI, HindIII sites of promoterless pBR322 derivative CAT plasmid, resulting in plasmid pBXΔ‐750.



Figure 17.

Promoter for RNA polymerase II containing separate sequence components with different roles. Sequences between components are not important. Orientation of conserved sequences is indicated by direction of arrows. Transcription factors bind to the conserved sequence motifs.

From Lewin 102a, reprinted by permission of John Wiley & Sons, Inc. © 1986


Figure 18.

Identification of DNA binding sites by DNase I footprinting.

From Lewin 102a, reprinted by permission of John Wiley & Sons, Inc. © 1986


Figure 19.

Splice commitment regulatory factor model for cell‐specific alternative RNA processing of a complex transcription unit in the brain. Putative factor that commits exon splicing pattern is represented by stippled oval and is postulated to inhibit usage of calcitonin poly(A) site.

From Leff et al. 99). Copyright 1987, by permission of Cell Press


Figure 20.

Nucleotide and amino acid sequence alignments of rat tumor cholecystokinin (CCK) and porcine gut gastrin (GSN) preprohormones. Amino acid identities are boxed. The putative initiator methionine in each case is designated +1. Deletions are indicated by dashed line.

From Deschenes et al. 34). Reprinted by permission of Elsevier Science Publishing Co., copyright 1985


Figure 21.

Schematic representation showing exon organization of human gastrin and rat cholecystokinin (CCK) genes. Second exons are aligned at putative initiator methionines, and third exons are aligned at conserved pentapeptide of preprohormones. Shaded boxes indicate translated exons, open boxes indicate nontranslated exons, and cross‐hatched boxes indicate conserved amino acids in preprohormones.



Figure 22.

Comparison of porcine amino acid sequence of neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PPP). Identities are underlined.



Figure 23.

Nucleotide sequence of pancreatic polypeptide cDNA clone. Restriction endonuclease sites for BamHI, HinfI, and AvaII, which were used for DNA sequencing, and oligonucleotide sequence corresponding to probe used for screening are indicated by solid lines. Deduced amino acid sequence is numbered from NH2‐terminal alanine of pancreatic polypeptide portion of sequence. Dashed lines, proposed leader sequence and icosapeptide portion; boxes, connecting sequence (Gly‐Lys‐Arg) and variant amino acid.

From Takeuchi and Yamada 182).


Figure 24.

Restriction map of human pancreatic polypeptide gene. Restriction sites in pPP4A subclone are shown. Four exons indicated as cross‐hatched boxes; repetitive DNA (Rep DNA) sequences shown as solid box and labeled; DNA sequencing strategy indicated by arrows below map; Solid arrows indicate 5'‐labeled fragments; and dashed arrows denote fragments labeled at 3’ ends base pairs (bp)

From Leiter et al. 101


Figure 25.

Nucleotide sequence encoding precursor to human NPY. Amino acid sequence of preproNPY numbered 1 through 97; termination codon denoted by asterisks; mature hormone is underlined; putative signal for poly(A) addition is underlined with broken line.

From Minth et al. 125


Figure 26.

Diagram of splicing of human neuropeptide Y (NPY) gene. Exons are represented as boxes, and dotted lines indicate how exons are spliced to produce mRNA; 5’ nontranslated sequences are located on exon 1 and shown as open box; second exon codes for signal peptide, residues 1–28, stippled box; mature NPY, residues 29–63, cross‐hatched box; third exon contains coding region for amino acid residues 64–90, solid box; fourth exon contains COOH‐terminal heptapeptide, residues 91–97, solid box; and 3’ nontranslated region, open box, including poly(A) addition site.

From Minth et al. 124


Figure 27.

Diagram of amino acid sequences and exon‐intron junctions of human neuropeptide Y (NPY) and human pancreatic polypeptide (PP). Boxes, identical amino acids. Splicing of gene after first two nucleotides of Arg codons depicted by splitting the three‐letter code for Arg after the first two letters (Ar‐g). Introns are represented by lines; their respective sizes are noted.

From Minth et al. 124


Figure 28.

Structures of two anglerfish preproglucagons 21,22 deduced from their respective cDNAs (AFG I and AFG II). Boxes, regions of protein sequence identity; arrows, prohormone processing sites.

From Lund et al. 109


Figure 29.

Structure of rat preproglucagon deduced from cDNA to rat preproglucagon. Circles, putative prohormone conversion sites. Identities of peptides indicated are NH2‐peptide, glicentin‐related polypeptide; IP‐I, intervening peptide‐I; IP‐II, intervening peptide‐II; GLP‐I, glucagon‐like peptide‐I; GLP‐II, glucagon‐like peptide‐II

From Heinrich et al. 72). Copyright 1984 by permission of The Endocrine Society


Figure 30.

Partial sequence of rat preproglucagon gene. Locations of exons indicated by capitalized bases and three‐letter code for deduced amino acids. Introns indicated by lowercase bases. Signal cleavage site (between −1 and +1) indicated by arrow, as are putative prohormone processing sites. Diagram at bottom summarizes exon‐intron structure of rat preproglucagon gene. Known or suspected bioactive peptides, solid boxes; nonhormone peptide portions, hatched boxes; signal peptide, stippled boxes; and noncoding portions of mRNA, open boxes.

From Heinrich et al. 71


Figure 31.

Structures of two related clones of human preproGRF. Bioactive regions enclosed by a light‐lined box; putative prohormone processing sites enclosed by dark‐lined boxes; differences between clones indicated by arrows; glycine that donates COOH‐terminal amide nitrogen indicated by asterisk; putative signal peptides underlined.

From Gubler et al. 66


Figure 32.

Sequence of human gene for preproGRF. Exons indicated by large characters.

From Mayo et al. 114
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Article Title: Molecular Biology of Gut Peptides
 

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Randy S. Haun, Carolyn D. Minth, Philip C. Andrews, Jack E. Dixon. Molecular Biology of Gut Peptides. Compr Physiol 2011, Supplement 17: Handbook of Physiology, The Gastrointestinal System, Neural and Endocrine Biology: 1-43. First published in print 1989. doi: 10.1002/cphy.cp060201