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

3. 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. Each restriction enzyme cleaves a specific sequence of bases called a restriction site.
These are often a symmetrical series of four to eight bases on both strands running in opposite directions.
If the restriction site on one strand is 3’-CTTAAG-5’, the complementary strand is 5’-GAATTC-3
Restriction enzymes cut covalent phosphodiester bonds of both strands, often in a staggered way 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 DNA vectors
Recombinant plasmids are produced by splicing restriction fragments from foreign DNA into plasmids.
A 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.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

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

11. The polymerase chain reaction (PCR) clones DNA entirely in vitro
When the source of DNA is scanty or impure, the polymerase chain reaction (PCR) is quicker and more selective. Its limitation is that PCR only 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.
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.
Fig. 20.7
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

13. PCR

14. This is faster than cloning via recombinant bacteria.
PCR can make billions of copies of a targeted DNA segment in a few hours.
This is faster than cloning via recombinant bacteria.
In PCR, a three-step cycle: heating, cooling, and replication, brings about a chain reaction that produces an exponentially growing population of DNA molecules.
PCR can make many copies of a specific gene before cloning in cells, simplifying the task of finding a clone with that gene.
PCR is so specific and powerful that only minute amounts of DNA need be present in the starting material
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

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. Chromosomal abnormalities
Incorrect number of chromosomes
Nondisjunction
chromosomes don’t separate properly during meiosis
Chromosome mutations
Deletion
Inversion
Duplication
Translocation

17. 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-1 n
n+1

18. Alteration of chromosome number

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

21. 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
but characteristic set of symptoms = syndrome

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

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

24. Karyotype of a normal male
Karyotyping
Chromosome Spread
Karyotype of a normal male

25. 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
Trisomy 13 occurs in about 1 out of every 5,000 live births. It is a syndrome with multiple abnormalities, many of which are not compatible with life. More than 80% of children with trisomy 13 die in the first month. Trisomy 13 is associated with multiple abnormalities, including defects of the brain that lead to seizures, apnea, deafness, and eye abnormalities. The eyes are small with defects in the iris (coloboma ). Most infants have a cleft lip and cleft palate, and low-set ears. Congenital heart disease is present in approximately 80% of affected infants. Hernias and genital abnormalities are common.
Trisomy 18 is a relatively common syndrome affecting approximately 1 out of 3,000 live births, and affecting girls more than three times as often as boys. The presence of an extra number 18 chromosome leads to multiple abnormalities. Many of these abnormalities make it hard for infants to live longer than a few months.
The cri du chat syndrome is caused by the deletion of information on chromosome 5. It is likely that multiple genes on chromosome 5 are deleted. One deleted gene, called TERT (telomerase reverse transcriptase) is involved in control of cell growth, and may play a role in how some of the features of cri cu chat develop. The cause of this rare chromosomal deletion is not known, but it is expected that the majority of cases are due to spontaneous loss of a piece of chromosome 5 during development of an egg or sperm. A minority of cases result from one parent carrying a rearrangement of chromosome 5 called a translocation. Between 1 in 20,000 and 1 in 50,000 babies are affected. This disease may account for up to 1% of individuals with severe mental retardation. Infants with cri du chat syndrome commonly have a distinctive cat-like cry. They also have an extensive grouping of abnormalities, with severe mental retardation being the most important.

26. Down syndrome & age of mother
Mother’s age
Incidence of Down Syndrome
Under 30
<1 in 1000 30
1 in 900 35
1 in 400 36
1 in 300 37
1 in 230 38
1 in 180 39
1 in 135 40
1 in 105 42
1 in 60 44
1 in 35 46
1 in 20 48
1 in 16 49
1 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

27. 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

28. Klinefelter’s syndrome
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

29. Klinefelter’s syndrome
How many Barr bodies would you expect?

30. 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

31. Trisomy X 3 X 1 in every 2000 live births produces healthy females
Why?
all but one X chromosome is inactivated
How many Barr bodies would you expect?

32. Turner syndrome 1X 1 in every 5000 births varied degree of effects
webbed neck
short stature
sterile
How many Barr bodies would you expect?

33. 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

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

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

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

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

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