1. Human Molecular Genetics Institute of Medical Genetics

3.  DefinitionDefinition  StructureStructure  OrganizationOrganization Outline of this chapter

5. Gene Molecular definition: DNA sequence encoding protein What are the problems with this definition?

6. Gene definition caveats  Some genomes are RNA instead of DNA  Some gene products are RNA (tRNA, rRNA, and others) instead of protein  Some nucleic acid sequences that do not encode gene products (noncoding regions) are necessary for production of the gene product (RNA or protein)

7.  Gene - is a segment of DNA encoding information leading to a functional product (RNA or polypeptide chain);  The most important feature of a gene is it must code for a functional product.  There are 30,000 to 35,000 genes in the human genome. Gene

8. Hybridization of mRNA and DNA Eukaryotic genes are split genes It includes coding region and noncoding regions.

9. A “Simple” Eukaryotic Gene Terminator Sequence Promoter/ Control Region Transcription Start Site RNA Transcript 5’ Untranslated Region 3’ Untranslated Region Exons Introns 3’5’ Exon 2Exon 3 Int. 2 Exon 1 Int. 1 3’5’ Exon 2Exon 3Exon 1 Int. 2Int. 1

10. Gene Structure  Exons Exons  IntronsIntrons  Regulatory sequencesRegulatory sequences - Promoter/proximal control elements - Enhancer/silencer - Terminator  Splicing junctionSplicing junction

11.  Segment of a gene which is decoded to give an mRNA product or a mature RNA product.  Individual exons may contain coding DNA or noncoding DNA (untranslated sequences, UTS).coding DNA Exons

12. Coding region Nucleotides (open reading frame) encoding the amino acid sequence of a protein

13.  Noncoding DNA which separates neighboring exons in a gene.  During gene expression introns, like exons, are transcribed into RNA but the transcribed intron sequences are subsequently removed by RNA splicing and are not present in mRNA. Introns

16.  Splice donor site: the junction between the end of an exon and the start of the downstream intron, commencing with the dinucleotide GT.  Splice acceptor site: the junction between the end of an intron terminating in the dinucleotide AG, and the start of the next exon.  Branch site: the third conserved intronic sequence that is known to be functionally important in splicing Splice junction (exon/intron boundary

17. Splice junction (exon/intron boundary

18. Splice junction (exon/intron boundary

19. Splice junction (exon/intron boundary)

20.  Consensus sequences are conserved throughout eukaryotes  Conservation of sequence is expected, since recognition of sequences is accomplished by base pairing with snRNPs RNA component Splice junction (exon/intron boundary)

21. Secondary structure model of human U1 snRNP. The region where it recognizes the pre- mRNA is also shown

22. Regulatory Sequences  5’ untranscribed region. Signals for initiation and control of transcription - Promoter/proximal elementsPromoter/proximal elements  Enhancer / Silencer Enhancer -Enhancer stimulates transcription -Silencer inhibits transcription  3’ untranscribed region. Signals for termination of transcriptionuntranscribed region

23. Regulatory Sequences

24. Promoter/Proximal Elements  Occur within ~200 bp of the start site.  Contain up to ~20 bp.  Cell-type specific

25. Basal Promoter Analysis  ATATAA-30TBP  GGCCAATC-75CTF/NF1  GCCACACCC-90SP1 TATACAATGC +1

26.  TATA box Most common Highly transcribed genes 25~35 base pairs upstream of start site  Initiator At start site  GC boxes (CpG islands) “Housekeeping” genes (transcribed at low rate) Within ~100 base pairs of start site Promoter-Proximal Elements

27. TATA box  ~ 25 bp upstream of +1  Only promoter element that is relatively fixed in relation to start point  Tends to be surrounded by GC-rich sequences  Single base substitutions in TATA  strong promoter down mutations  Some promoters do not contain TATA

28. Initiator  Instead of a TATA box, some eukaryotic gene contain an alternative promoter element, called an initiator.  Initiator is highly degenerative. 5’ Y Y A N T/A Y Y Y +1 Y = pyrimidine (C or T) N = any

29. CpG island  Genes coding for intermediary metabolism are transcribed at low rates, and do not contain a TATA box or initiator.  Most genes of this type contain a CG-rich stretch of 20-50 nt within ~100 bp upstream of the start site region.  A transcription factor called SP1 recognizes these CG-rich region.  Gives multiple alternative mRNA start sites. mRNA Multiple 5’-start sites CpG island ~100 bp

30. Enhancers  Can be located several kb from promoter  Can be present in either orientation relative to the promoter  Contain elements that bind inducible factors  Usually ~100-200 bp long, containing multiple 8- to 20-bp control elements.  Targets for tissue specific and/or temporal regulation

31. Enhancer  Variable distance from promoter  Either orientation  Upstream or downstream of gene

32. TERMINATION RNA polymerase meets the terminator Terminator sequence: AAUAAA RNA polymerase releases from DNA Prokaryotes-releases at termination signal Eukaryotes-releases 10-35 base pairs after termination signal

33. Termination  Different mechanisms of termination  Prokaryotes  rho-independent termination: formation of a hairpin structure  rho-dependent termination: external protein disrupts transcription  Eukaryotes  cleavage of the RNA by an external protein

34. Rho-independent terminator

35. Distribution  Different density of genes along a chromosome  Different density of genes between chromosomes

37. (exon-intron-exon) n structure of various genes  -globin HGPRT (HPRT) total = 1,660 bp; exons = 990 bp histone factor VIII total = 400 bp; exon = 400 bp total = 42,830 bp; exons = 1263 bp total = ~186,000 bp; exons = ~9,000 bp

39. Genes  Protein Coding  RNA genes  rRNA  tRNA  snRNA, snoRNA …

40. ”Average” gene organization  Single, unique genes consisting of exons interrupted by introns only

41. Other gene organizations  Dispersed gene segments brought together by genome reorganization in specialized cells  Example: gene for  T-cell receptor protein in T-cells

42. Light Chain Gene Families Germ line gene organization J1J1 C1C1 E J2J2 C2C2 E J3J3 C3C3 E J4J4 C4C4 E P L V1V1 P L VnVn P L V2V2 Lambda light chain genes; n=30 P L V  1 P L V  n P L V  2 J2J2J3J3J5J5 CC E J1J1J4J4 Kappa light chain genes; n=300

43. Light Chain Gene Families Gene rearrangement and expression L V 1V 1 L V nV n L V 2V 2 J2J2J3J3J5J5 CC J1J1J4J4 DNA PPP E P P E L V 1V 1 L J5J5 CC V 2V 2J4J4 DNA Rearrangement VC J V C Protein Transport to ER Protein Translation C L V J E L V CJ RNA Transcription Primary transcript C L V J RNA Processing RNA mRNA

44. Heavy Chain Gene Family Germ line gene organization

45. Heavy Chain Gene Family Gene rearrangement and expression P L V1 P L Vn P L V2 D2D2 D3D3 D1D1DnDn CC CC J2J2J3J3J5J5J1J1J4J4 E DJ rearrangement P L Vn P L V1 P L V2 D2D2 D1D1 CC CC J5J5 J4J4 E DNA VDJ rearrangement D2D2 CC CC J5J5 J4J4 E P L V1V1 P L V2V2 DNA Transcription D2D2 CC CC J5J5 J4J4 E L V2V2 RNA Primary transcript

46. Other gene organizations

47.  Genes-within-genes  It is not uncommon that short genes are located inside an intron of another gene

48. Intron 26 of the NF1 gene contains three internal genes.

49. Other gene organizations  Gene families: functionally similar or identical genes repeated on the same or different chromosomes  Example 1: genes for histones and (ribosomal) rRNA  Example 2: The globin families

50.  Gene families defined by conserved amino acid motifs  DEAD box.  WD repeat families

51.  Clustered gene families Growth hormone5 copies (67kb)  globin7 copies (50kb) Hox genes (multi) 38 four clusters Olfactory receptors1000 in 25 large clusters

53.  Interspersed gene families Pax9 copies Actin>20 copies Alu elements (repeats)1.1 million LINE elements (L1)200-500,000

55. Pseudogenes  Nonfunctional copies of genes  Formed by duplication of ancestral gene, or reverse transcription (and integration)  Not expressed due to mutations that produce a stop codon (nonsense or frameshift) or prevent mRNA processing, or due to lack of regulatory sequences