1. CHAPTER 15 LECTURE SLIDES
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2. Genes and How They Work
Chapter 15

3. The Nature of Genes
Early ideas to explain how genes work came from studying human diseases
Archibald Garrod – 1902
Recognized that alkaptonuria is inherited via a recessive allele
Proposed that patients with the disease lacked a particular enzyme
These ideas connected genes to enzymes

4. Beadle and Tatum – 1941
Deliberately set out to create mutations in chromosomes and verify that they behaved in a Mendelian fashion in crosses
Studied Neurospora crassa
Used X-rays to damage DNA
Looked for nutritional mutations
Had to have minimal media supplemented to grow

5. Beadle and Tatum looked for fungal cells lacking specific enzymes
The enzymes were required for the biochemical pathway producing the amino acid arginine
They identified mutants deficient in each enzyme of the pathway
One-gene/one-enzyme hypothesis has been modified to one-gene/one-polypeptide hypothesis

7. Central Dogma First described by Francis Crick
Information only flows from
DNA → RNA → protein
Transcription = DNA → RNA
Translation = RNA → protein
Retroviruses violate this order using reverse transcriptase to convert their RNA genome into DNA

9. Transcription Translation DNA-directed synthesis of RNA
Only template strand of DNA used
U (uracil) in DNA replaced by T (thymine) in RNA
mRNA used to direct synthesis of polypeptides
Translation
Synthesis of polypeptides
Takes place at ribosome
Requires several kinds of RNA

10. RNA All synthesized from DNA template by transcription
Messenger RNA (mRNA)
Ribosomal RNA (rRNA)
Transfer RNA (tRNA)
Small nuclear RNA (snRNA)
Signal recognition particle RNA
Micro-RNA (miRNA)

11. Genetic Code
Francis Crick and Sydney Brenner determined how the order of nucleotides in DNA encoded amino acid order
Codon – block of 3 DNA nucleotides corresponding to an amino acid
Introduced single nulcleotide insertions or deletions and looked for mutations
Frameshift mutations
Indicates importance of reading frame

13. Marshall Nirenberg identified the codons that specify each amino acid
Stop codons
3 codons (UUA, UGA, UAG) used to terminate translation
Start codon
Codon (AUG) used to signify the start of translation
Code is degenerate, meaning that some amino acids are specified by more than one codon

15. Code practically universal
Strongest evidence that all living things share common ancestry
Advances in genetic engineering
Mitochondria and chloroplasts have some differences in “stop” signals

16. Prokaryotic transcription
Single RNA polymerase
Initiation of mRNA synthesis does not require a primer
Requires
Promoter
Start site Transcription unit
Termination site

17. Promoter Forms a recognition and binding site for the RNA polymerase
Found upstream of the start site
Not transcribed
Asymmetrical – indicate site of initiation and direction of transcription

18. a.   Holoenzyme Core enzyme  ׳  Prokaryotic RNA polymerase 18
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Holoenzyme
Core
enzyme  ׳ 
Prokaryotic RNA polymerase a. 18

19. 19  TATAAT– Promoter (–10 sequence)  Holoenzyme Core enzyme 
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TATAAT– Promoter (–10 sequence) 
Holoenzyme
Core
enzyme 
Downstream 5׳ 9 3׳ 
Start site (+1)
Template
strand
TTGACA–Promoter
(–35 sequence)
Coding
strand
Prokaryotic RNA polymerase
Upstream 5׳ 3׳ a. b. 19

20. Copyright © The McGraw-Hill Companies, Inc
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 binds to DNA 
TATAAT– Promoter (– 10 sequence) 
Core
enzyme
Holoenzyme 
Downstream 5׳ 5׳ 9 3׳ 3׳ 
Start site (+1)
Template
strand
TTGACA – Promoter
(–35 sequence)
Helix opens at
– 1 0 se q uence
Coding
strand
Prokaryotic RNA polymerase
Upstream 5׳ 3׳ 5׳ 3׳ a. b. 20

21. 21  TATAAT– Promoter (–10 sequence)  Holoenzyme Core enzyme 
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 
TATAAT– Promoter (–10 sequence) 
Holoenzyme
Core
enzyme 
Downstream 5׳ 9 3׳ 
Start site (+1)
Template
strand
TTGACA–Promoter
(–35 sequence)
Coding
strand
Prokaryotic RNA polymerase
Upstream 5׳ 3׳ a. b.
 binds to DNA
RNA polymerase bound
to unwound DNA
Transcription
bubble 5׳ 3׳
 dissociates
ATP
Helix opens at
–10 sequence
Start site RNA
synthesis begins 5׳ 3׳ 21

22. Elongation
Grows in the 5′-to-3′ direction as ribonucleotides are added
Transcription bubble – contains RNA polymerase, DNA template, and growing RNA transcript
After the transcription bubble passes, the now-transcribed DNA is rewound as it leaves the bubble

24. Termination Marked by sequence that signals “stop” to polymerase
Causes the formation of phosphodiester bonds to cease
RNA–DNA hybrid within the transcription bubble dissociates
RNA polymerase releases the DNA
DNA rewinds
Hairpin

26. Prokaryotic transcription is coupled to translation
mRNA begins to be translated before transcription is finished
Operon
Grouping of functionally related genes
Multiple enzymes for a pathway
Can be regulated together

28. Eukaryotic Transcription
3 different RNA polymerases
RNA polymerase I transcribes rRNA
RNA polymerase II transcribes mRNA and some snRNA
RNA polymerase III transcribes tRNA and some other small RNAs
Each RNA polymerase recognizes its own promoter

29. Initiation of transcription
Requires a series of transcription factors
Necessary to get the RNA polymerase II enzyme to a promoter and to initiate gene expression
Interact with RNA polymerase to form initiation complex at promoter
Termination
Termination sites not as well defined

30. Other transcription factors
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Other transcription factors
Eukaryotic
DNA
Transcription
factor
TATA box

31. Other transcription factors RNA polymerase II
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Other transcription factors
RNA polymerase II
Eukaryotic
DN A
Transcription
factor
TATA box 31

32. 32 Other transcription factors RNA polymerase II Eukaryotic DNA
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Other transcription factors
RNA polymerase II
Eukaryotic
DNA
Transcription
factor
Initiation
complex
TATA box 32

33. mRNA modifications
In eukaryotes, the primary transcript must be modified to become mature mRNA
Addition of a 5′ cap
Protects from degradation; involved in translation initiation
Addition of a 3′ poly-A tail
Created by poly-A polymerase; protection from degradation
Removal of non-coding sequences (introns)
Pre-mRNA splicing done by spliceosome

34. 5´ cap HO OH P P P CH2 + 3´ poly-A tail 3´ N+ A A A A A A A CH3
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5´ cap HO OH P P P
CH2 +
3´ poly-A tail 3´ N+
A A A A A A A
CH3
Methyl group
A A U A A A
mRNA P P P G 5´
CH3

35. Eukaryotic pre-mRNA splicing
Introns – non-coding sequences
Exons – sequences that will be translated
Small ribonucleoprotein particles (snRNPs) recognize the intron–exon boundaries
snRNPs cluster with other proteins to form spliceosome
Responsible for removing introns

36. a. E1 I1 E 2 I 2 E3 I3 E 4 I 4 DNA template Transcription 5׳ cap
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E 2
I 2 E3 I3
E 4
I 4
DNA template
Exons
Transcription
Introns
5׳ cap
33׳ poly-A tail
Primary RNA transcript
Introns are removed
5׳ cap
3׳ poly-A tail a.
Mature mRNA

37. b: Courtesy of Dr. Bert O’Malley, Baylor College of Medicine
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. E1 I1
E 2
I 2 E3 I3
E 4
I 4
DNA template
Exons
Transcription
Introns
5׳ cap
3׳ poly-A tail
Primary RNA transcript
Introns are removed
5׳ cap
3׳ poly-A tail a.
Mature mRNA
Intron 1
mRNA 3 2 4
DNA 7 5 6
Exon b. c. 37
b: Courtesy of Dr. Bert O’Malley, Baylor College of Medicine

38. 1. snRNA forms base-pairs with 5´ end of intron, and at branch site.
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snRNA
snRNPs
Exon 1
Intron
Exon 2 A 5´ 3´
Branch point A
1. snRNA forms base-pairs with 5´ end of intron, and at branch site.

39. 1. snRNA forms base-pairs with 5´ end of intron, and at branch site.
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snRNA
snRNPs
Exon 1
Intron
Exon 2 A 5´ 3´
Branch point A
1. snRNA forms base-pairs with 5´ end of intron, and at branch site.
Spliceosome A 5´ 3´
2. snRNPs associate with other factors to form spliceosome. 39

40. 40 snRNA snRNPs Exon 1 Intron Exon 2 A 5´ 3´ Branch point A
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snRNA
snRNPs
Exon 1
Intron
Exon 2 A 5´ 3´
Branch point A
1. snRNA forms base-pairs with 5´ end of intron, and at branch site.
Spliceosome A 5´ 3´
2. snRNPs associate with other factors to form spliceosome.
Lariat A 5´ 3´
3. 5´ end of intron is removed and forms bond at branch site,
forming a lariat. The 3´ end of the intron is then cut. 40

41. 41 snRNA snRNPs Exon 1 Intron Exon 2 A 5´ 3´ Branch point A
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snRNA
snRNPs
Exon 1
Intron
Exon 2 A 5´ 3´
Branch point A
1. snRNA forms base-pairs with 5´ end of intron, and at branch site.
Spliceosome A 5´ 3´
2. snRNPs associate with other factors to form spliceosome.
Lariat A 5´ 3´
3. 5´ end of intron is removed and forms bond at branch site,
forming a lariat. The 3´ end of the intron is then cut.
Excised
intron
Exon 1
Exon 2 5´
M ature mRNA 3´ 41
4. Exons are joined; spliceosome disassembles.

42. Alternative splicing
Single primary transcript can be spliced into different mRNAs by the inclusion of different sets of exons
15% of known human genetic disorders are due to altered splicing
35 to 59% of human genes exhibit some form of alternative splicing
Explains how 25,000 genes of the human genome can encode the more than 80,000 different mRNAs

43. tRNA and Ribosomes
tRNA molecules carry amino acids to the ribosome for incorporation into a polypeptide
Aminoacyl-tRNA synthetases add amino acids to the acceptor stem of tRNA
Anticodon loop contains 3 nucleotides complementary to mRNA codons

44. 2D “Cloverleaf” Model 3D Ribbon-like Model 3D Space-filled Model Icon
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2D “Cloverleaf” Model
3D Ribbon-like Model
Acceptor end
Acceptor end 3׳ 5׳
Anticodon
loop
Anticodon loop
3D Space-filled Model
Icon
Acceptor end
Acceptor end
Anticodon loop
Anticodon end
c: Created by John Beaver using ProteinWorkshop, a product of the RCSB PDB, and built using the Molecular Biology Toolkit developed by John Moreland and Apostol Gramada (mbt.sdsc.edu). The MBT is fi nanced by grant GM63208

45. tRNA charging reaction
Each aminoacyl-tRNA synthetase recognizes only 1 amino acid but several tRNAs
Charged tRNA – has an amino acid added using the energy from ATP
Can undergo peptide bond formation without additional energy
Ribosomes do not verify amino acid attached to tRNA

47. Pi Pi Amino group Carboxyl group NH3+ Trp C O O– ATP Amino acid site
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Amino
group
Carboxyl
group
NH3+
Trp C O O–
ATP
Amino
acid site Pi Pi
tRNA
site
Aminoacyl-tRNA
synthetase

48. Pi Pi Amino group Carboxyl group NH3+ NH3+ NH3+ Trp C O Trp Trp C O– O
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Amino
group
Carboxyl
group
NH3+
NH3+
NH3+
Trp C O
Trp
Trp C O– O C
ATP
Accepting
site
AMP O
AMP O
Amino
acid site O OH Pi Pi OH
tRNA
tRNA
site
Aminoacyl-tRNA
synthetase
Anticodon
specific to tryptophan 48

49. 49 Pi Pi Amino group Carboxyl group Charged tRNA travels to ribosome
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Amino
group
Carboxyl
group
Charged tRNA travels to ribosome
NH3+
NH3+
Trp C
NH3+ O
Trp C
Trp
NH3+
Trp C O O– O C
NH3+
ATP
Accepting
site
AMP
AMP O O
Trp O
Amino
acid site O C
AMP O Pi Pi OH OH O
tRNA
tRNA
site
Aminoacyl-tRNA
synthetase
Charged
tRNA
dissociates
Anticodon
specific to tryptophan 49

50. The ribosome has multiple tRNA binding sites
P site – binds the tRNA attached to the growing peptide chain
A site – binds the tRNA carrying the next amino acid
E site – binds the tRNA that carried the last amino acid

51. Large subunit 3´ 90 ° Small subunit Large subunit Small subunit Large
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Large
subunit 3´ 90
Small
subunit
Large
subunit
Small
subunit
Large
subunit
Small
subunit
mRNA 5´

52. The ribosome has two primary functions
Decode the mRNA
Form peptide bonds
Peptidyl transferase
Enzymatic component of the ribosome
Forms peptide bonds between amino acids

53. Translation In prokaryotes, initiation complex includes
Initiator tRNA charged with N-formylmethionine
Small ribosomal subunit
mRNA strand
Ribosome binding sequence (RBS) of mRNA positions small subunit correctly
Large subunit now added
Initiator tRNA bound to P site with A site empty

55. Initiations in eukaryotes similar except
Initiating amino acid is methionine
More complicated initiation complex
Lack of an RBS – small subunit binds to 5′ cap of mRNA

56. Elongation adds amino acids
2nd charged tRNA can bind to empty A site
Requires elongation factor called EF-Tu to bind to tRNA and GTP
Peptide bond can then form
Addition of successive amino acids occurs as a cycle

59. There are fewer tRNAs than codons
Wobble pairing allows less stringent pairing between the 3′ base of the codon and the 5′ base of the anticodon
This allows fewer tRNAs to accommodate all codons

60. Termination
Elongation continues until the ribosome encounters a stop codon
Stop codons are recognized by release factors which release the polypeptide from the ribosome

62. Protein targeting
In eukaryotes, translation may occur in the cytoplasm or the rough endoplasmic reticulum (RER)
Signal sequences at the beginning of the polypeptide sequence bind to the signal recognition particle (SRP)
The signal sequence and SRP are recognized by RER receptor proteins
Docking holds ribosome to RER
Beginning of the protein-trafficking pathway

64. Primary RNA transcript Primary RNA transcript
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RNA polymerase II
RNA polymerase II
1. RNA polymerase
II in the nucleus
copies one
strand
of the DNA to
produce the
primary
transcript. 3´ 3´ 5´ 5´
Primary RNA transcript
Primary RNA transcript

65. 65 RNA polymerase II RNA polymerase II 1. RNA polymerase
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RNA polymerase II
RNA polymerase II
1. RNA polymerase
II in the nucleus
copies one
strand
of the DNA to
produce the
primary
transcript. 3´ 3´
Primary RNA transcript
Primary RNA transcript
2. The primary transcript
is processed by
addition of a 5´
methyl-G cap,
cleavage and
polyadenylation of the
3´ end, and removal of
introns. The mature
mRNA is then
exported through
nuclear pores to the
cytoplasm.
Poly-A tail
Poly-A tail 5´ 5´
Primary RNA transcript
Primary RNA transcript
Cut intron
Cut intron
Mature mRNA
Mature mRNA
5´ cap
5´ cap 65

66. Copyright © The McGraw-Hill Companies, Inc
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RNA polymerase II
RNA polymerase II
1. RNA polymerase
II in the nucleus
copies one
strand
of the DNA to
produce the
primary
transcript. 3´ 3´
Primary RNA transcript
Primary RNA transcript
2. The primary transcript
is processed by
addition of a 5´
methyl-G cap,
cleavage and
polyadenylation of the
3´ end, and removal of
introns. The mature
mRNA is then
exported through
nuclear pores to the
cytoplasm.
Poly-A tail
Poly-A tail 5´ 5´
Primary RNA transcript
Primary RNA transcript
Cut intron
Cut intron
Mature mRNA
Mature mRNA
5´ cap
5´ cap
3. The 5´ cap of the
mRNA
associates with
the small subunit
of the ribosome.
The initiator
tRNA and large
subunit are
added to form
an initiation
complex.
Large
subunit
5´ cap
mRNA
Small
subunit
Cytoplasm 66

67. Copyright © The McGraw-Hill Companies, Inc
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RNA polymerase II
RNA polymerase II
1. RNA polymerase
II in the nucleus
copies one
strand
of the DNA to
produce the
primary
transcript. 3´ 3´
Primary RNA transcript
Primary RNA transcript
2. The primary transcript
is processed by
addition of a 5´
methyl-G cap,
cleavage and
polyadenylation of the
3´ end, and removal of
introns. The mature
mRNA is then
exported through
nuclear pores to the
cytoplasm.
Poly-A tail
Poly-A tail 5´ 5´
Primary RNA transcript
Primary RNA transcript
Cut intron
Cut intron
Mature mRNA
Mature mRNA
5´ cap
5´ cap
3. The 5´ cap of the
mRNA
associates with
the small subunit
of the ribosome.
The initiator
tRNA and large
subunit are
added to form
an initiation
complex.
Large
subunit
5´ cap
mRNA
Small
subunit
Cytoplasm
Cytoplasm
Amino acids
tRNA arrivesin A site 3´
mRNA
A site
P site 5´
E site
4. The ribosome cycle begins with the
growing peptide attached to the tRNA
in the P site. The next charged tRNA
binds to the A site with its anticodon
complementary to the codon in the
mRNA in this site. 67

68. Copyright © The McGraw-Hill Companies, Inc
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RNA polymerase II
RNA polymerase II
1. RNA polymerase
II in the nucleus
copies one
strand
of the DNA to
produce the
primary
transcript. 3´ 3´
Primary RNA transcript
Primary RNA transcript
2. The primary transcript
is processed by
addition of a 5´
methyl-G cap,
cleavage and
polyadenylation of the
3´ end, and removal of
introns. The mature
mRNA is then
exported through
nuclear pores to the
cytoplasm.
Poly-A tail
Poly-A tail 5´ 5´
Primary RNA transcript
Primary RNA transcript
Cut intron
Cut intron
Mature mRNA
Mature mRNA
5´ cap
5´ cap
3. The 5´ cap of the
mRNA
associates with
the small subunit
of the ribosome.
The initiator
tRNA and large
subunit are
added to form
an initiation
complex.
Large
subunit
5´ cap
mRNA
Small
subunit
Cytoplasm
Cytoplasm
Lengthening
polypeptide chain
Amino acids
tRNA arrivesin A site 3´
Emptyt
RNA 3´
mRNA
A site
P site 5´
E site 5´
4. The ribosome cycle begins with the
growing peptide attached to the tRNA
in the P site. The next charged tRNA
binds to the A site with its anticodon
complementary to the codon in the
mRNA in this site.
5. Peptide bonds form between the
amino terminus of the next amino
acid and the carboxyl terminus of
the growing peptide. This transfers
the growing peptide to the tRNA in
the A site, leaving the tRNA in the
P site empty. 68

69. Copyright © The McGraw-Hill Companies, Inc
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RNA polymerase II
RNA polymerase II
1. RNA polymerase
II in the nucleus
copies one
strand
of the DNA to
produce the
primary
transcript. 3´ 3´
Primary RNA transcript
Primary RNA transcript
2. The primary transcript
is processed by
addition of a 5´
methyl-G cap,
cleavage and
polyadenylation of the
3´ end, and removal of
introns. The mature
mRNA is then
exported through
nuclear pores to the
cytoplasm.
Poly-A tail
Poly-A tail 5´ 5´
Primary RNA transcript
Primary RNA transcript
Cut intron
Cut intron
Mature mRNA
Mature mRNA
5´ cap
5´ cap
3. The 5´ cap of the
mRNA
associates with
the small subunit
of the ribosome.
The initiator
tRNA and large
subunit are
added to form
an initiation
complex.
Large
subunit
5´ cap
mRNA
Small
subunit
Cytoplasm
Cytoplasm
Empty tRNA moves into
E site and is ejected
Lengthening
polypeptide chain
Amino acids
tRNA arrivesin A site 3´
Emptyt
RNA 3´ 3´
mRNA
A site
P site 5´
E site 5´ 5´
4. The ribosome cycle begins with the
growing peptide attached to the tRNA
in the P site. The next charged tRNA
binds to the A site with its anticodon
complementary to the codon in the
mRNA in this site.
5. Peptide bonds form between the
amino terminus of the next amino
acid and the carboxyl terminus of
the growing peptide. This transfers
the growing peptide to the tRNA in
the A site, leaving the tRNA in the
P site empty.
6. Ribosome translocation moves the
ribosome relative to the mRNA and
its bound tRNAs. This moves the
growing chain into the P site, leaving
the empty tRNA in the E site and the
A site ready to bind the next
charged tRNA. 69

71. Mutation: Altered Genes
Point mutations alter a single base
Base substitution – substitute one base for another
Silent mutation – same amino acid inserted
Missense mutation – changes amino acid inserted
Transitions
Transversions
Nonsense mutations – changed to stop codon

74. Nonpolar (hydrophobic)
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Normal H B B Sequence
Normal
Deoxygenated
Tetramer
Abnormal
Deoxygenated
Tetramer
Polar
Leu
Thr
Pro
Glu
Glu
Lys
Ser
Amino acids 1 2 1 2 1 2 1 2 C T G A C T C C T G A G A A G A A G T C T
Nucleotides
Hemoglobin
tetramer
"Sticky" non-
polar sites
Abormal H B B Sequence
Nonpolar (hydrophobic)
Leu
Thr
Pro
val
Glu
Lys
Ser
Amino acids
Tetramers form long chains
when deoxygenated. This
distorts the normal red blood
cell shape into a sickle shape. C T G A C T C C T G T G A A G A A G T C T
Nucleotides

75. Frameshift mutations Addition or deletion of a single base
Much more profound consequences
Alter reading frame downstream
Triplet repeat expansion mutation
Huntington disease
Repeat unit is expanded in the disease allele relative to the normal

76. Chromosomal mutations
Change the structure of a chromosome
Deletions – part of chromosome is lost
Duplication – part of chromosome is copied
Inversion – part of chromosome in reverse order
Translocation – part of chromosome is moved to a new location

79. Mutations are the starting point for evolution
Too much change, however, is harmful to the individual with a greatly altered genome
Balance must exist between amount of new variation and health of species