1. Green with envy?? Jelly fish “GFP” Transformed vertebrates

3. DNA Manipulation, Gene Cloning and Karyotyping

4. Gene Cloning Techniques for gene cloning enable scientists to prepare multiple identical copies of gene- sized pieces of DNA. Most methods for cloning pieces of DNA share certain general features. –For example, a foreign gene is inserted into a bacterial plasmid and this recombinant DNA molecule is returned to a bacterial cell. –Every time this cell reproduces, the recombinant plasmid is replicated as well and passed on to its descendents. –Under suitable conditions, the bacterial clone will make the protein encoded by the foreign gene.

5. One goal may be to produce a protein product for use. A second goal may be to prepare many copies of the gene itself. –This may enable scientists to determine the gene’s nucleotide sequence or provide an organism with a new metabolic capability by transferring a gene from another organism.

6. Restriction Enzymes In nature, bacteria use restriction enzymes to cut foreign DNA, such as from phages or other bacteria. Most restrictions enzymes are very specific, recognizing short DNA nucleotide sequences and cutting at specific point in these sequences.

7. Restriction enzyme cleaves DNA at specific sequence of bases called a restriction site. –often a symmetrical series of four to eight bases on both strands running in opposite directions. –restriction site on one strand is 3’-CTTAAG-5’, the complementary strand is 5’-GAATTC-3 Restriction enzymes cut the sugar phosphate backbone bonds of both DNA strands, creating single-stranded ends, sticky ends. –These extensions will form hydrogen-bonded base pairs with complementary single-stranded stretches on other DNA molecules cut with the same restriction enzyme

9. Recombinant plasmids--produced by splicing restriction fragments from foreign DNA into plasmids. –Plasmid is a circular piece of DNA found in bacteria and contain genes. –Plasmids can be used to insert DNA from another organism into a bacterial cell. Then, as a bacterium carrying a recombinant plasmid reproduces, the plasmid replicates within it. Recombinant DNA vectors Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

10. Cloning a gene into a bacterial plasmid can be divided into five steps. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings Fig. 20.3 Blue colonies White colonies

11. When the source of DNA is small or impure, the polymerase chain reaction (PCR) is quicker and more selective. (limitation of PCR -- produces short DNA segments within a gene and not entire genes.) This technique can quickly amplify any piece of DNA without using cells. Devised in 1985, PCR has had a major impact on biological research and technology. The polymerase chain reaction (PCR) clones DNA entirely in vitro Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

12. The DNA is incubated in a test tube with special DNA polymerase, a supply of nucleotides, and short pieces of single- stranded DNA as a primer. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings Fig. 20.7

13. PCR

14. PCR can make billions of copies of a targeted DNA segment in a few hours. –This is faster than cloning via recombinant bacteria. PCR, a three-step cycle: –heating, –cooling, –replication, –brings about a chain reaction that produces an exponential number of DNA molecules. PCR is so specific and powerful that only minute amounts of DNA need be present in the starting material

15. PCR has amplified DNA from a variety of sources: –fragments of ancient DNA from a 40,000-year- old frozen wooly mammoth, –DNA from tiny amount of blood or semen found at the scenes of violent crimes, –DNA from single embryonic cells for rapid prenatal diagnosis of genetic disorders, –DNA of viral genes from cells infected with difficult-to-detect viruses such as HIV. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

16. Human chromosome disorders High frequency in humans –most embryos are spontaneously aborted –alterations are too disastrous –developmental problems result from biochemical imbalance imbalance in regulatory molecules? –hormones? –transcription factors? Certain conditions are tolerated –upset the balance less = survivable –characteristic set of symptoms = syndrome

17. Chromosomal abnormalities Incorrect numbers of chromosomes(Numerical) –Nondisjunction chromosomes don’t separate properly during meiosis Structural abnormalities –Chromosome mutations Deletion Inversion Duplication Translocation

18. Numerical Abnormalities: Nondisjunction Problems with meiotic spindle cause errors in daughter cells –homologous chromosomes do not separate properly during Meiosis 1 –sister chromatids fail to separate during Meiosis 2 –too many or too few chromosomes 2n n n n-1 n+1

19. Alteration of chromosome number

20. Nondisjunction Baby has wrong chromosome number –Trisomy cells have 3 copies of a chromosome –Monosomy cells have only 1 copy of a chromosome n+1n n-1n monosomy 2n-1 trisomy 2n+1

22. Down syndrome Trisomy 21 –3 copies of chromosome 21 –1 in 700 children born in U.S. Chromosome 21 is the smallest human chromosome –but still severe effects Frequency of Down syndrome correlates with the age of the mother

23. Down syndrome & age of mother Mother’s age Incidence of Down Syndrome Under 30<1 in 1000 301 in 900 351 in 400 361 in 300 371 in 230 381 in 180 391 in 135 401 in 105 421 in 60 441 in 35 461 in 20 481 in 16 491 in 12 Rate of miscarriage due to amniocentesis:  1970s data 0.5%, or 1 in 200 pregnancies  2006 data <0.1%, or 1 in 1600 pregnancies

24. Sex chromosomes abnormalities Human development more tolerant of wrong numbers in sex chromosome But produces a variety of distinct syndromes in humans –XXY = Klinefelter’s syndrome male –XXX = Trisomy X female –XYY = Jacob’s syndrome male –XO = Turner syndrome female

25. 2X and 1Y –one in every 2000 live births –have male sex organs, but are sterile –feminine characteristics some breast development lack of facial hair –tall –normal intelligence Klinefelter’s syndrome

27. Jacob’s syndrome male 1X and 2 Y –1 in 1000 live male births –extra Y chromosome –slightly taller than average –more active –normal intelligence, slight learning disabilities –delayed emotional immaturity –normal sexual development

28. Trisomy X 3 X –1 in every 2000 live births –produces healthy females Why? all but one X chromosome is inactivated

29. Turner syndrome 1X –1 in every 5000 births –varied degree of effects –webbed neck –short stature –sterile

30. Changes in chromosome structure Deletion –loss of a chromosomal segment Duplication –repeat a segment Inversion –reverses a segment Translocation –move segment from one chromosome to another error of replication error of crossing over

31. 46,XY,t(8;9)(q24.3;q22.1) Translocation

32. FISH analysis: abl/bcr Genes on Diploid Cells and Ph Positive CML Cells Normal Translocation

33. Genetics Laboratory Cytogenetics Tissue culture Harvesting/Slide Preparation Karyotyping Results / Interpretation Report FISH Analysis

34. Genetic testing Amniocentesis in 2nd trimester –sample of embryo cells –stain & photograph chromosomes Analysis of karyotype

35. Karyotyping Karyotype of a normal male Chromosome Spread

36. Dosage Compensation Do males have half as much of the products of genes on the X as do females? NO!!

37. X Inactivation Interphase: Chromomes can’t be stained, but a dark-staining body is visible in the nuclei of cells of female mammals Barr Body: Inactive X

38. Which X gets inactivated? Mary Lyon & Lianne Russell (1961) proposed that one or other of X becomes inactivated at a particular time in early development. Within each cell,which X becomes inactivated is random. As development proceeds, all cells arising by cell division after that time have same X inactivated.

39. In 64-cell embryos Adult female mammals have two copies of each gene on the X chromosome.