1. Organization of genes on chromosomes
Mapping genes on chromosome:
Genetic mapping (linkage and recombination analyses).
Cytogenetics method.
Restriction mapping, deletion studies, and other molecular approaches such as chromosome walking.
Sequencing.
Deduction of gene structure.

3. Restriction Mapping

5. Restriction Fragment Length Polymorphism (RFLP)
RFLP and VNTR (variable lengths of tandem repeats) are
useful in DNA fingerprinting.

6. Locating gene by chromosomal walking.

7. A case with the Duchenne muscular dystrophy gene.

8. Deduction of gene structure
By hybridization of mRNA and chromosomal DNA.
By restriction mapping.
By DNA sequence.

9. Eukaryotic genes are often interrupted

11. Exons and introns

12. Organization of interrupted genes may be deduced by
restriction mapping of cDNA and genomic DNA
Small exons or introns may be missed by such an analysis.
Resolution at the sequence level is necessary to identify all
segments of the gene.

13. Figure 4.12: Exons are identified by flanking sequences and ORFs.

14. Introns of nuclear genes generally have termination codons
in all reading frames, and have no coding function.

15. Organization of interrupted genes may be conserved: all
globin genes have the following interrupted structures.

16. Another example with the DHFR gene.
Exon sequences are conserved but introns vary

17. Frequency of interrupted genes in some eukaryotes

19. Exons are usually short

21. Some DNA sequences code for more than one protein

22. Two genes may share the same sequence by reading the DNA
in different frames

23. Alternative splicing of mRNA can produce more than one protein

24. Genes can be identified and isolated by many approaches
By classical genetic approach.
By reverse genetic approach.
DNA cloning and/or cDNA approach.
By conservation of exons (eg., zoo blotting).
By analysis of DNA sequence (ORF flanked by splicing junction).
By exon trapping.
By chromosome walking (especially for large-sized gene).

25. How would you know that a segment of DNA is part of a gene?
Cross-hybridization with
the genomes of other
species (Zoo blot).
2. Contains open reading
frames (ORF).

26. Characterization of dystrophin gene by zoo blotting,
cDNA identification and chromosomal walking.

27. Exon trapping

28. How did interrupted genes evolve?
Introns early or introns late?
1. The equation of at least some exons with protein domains, and the appearance of related exons in different proteins, indicates that the duplication and juxtaposition of exons has played an important role in evolution.
2. Most protein-coding genes probably originated in an interrupted form, but interrupted genes that code for RNA have originally been uninterrupted.

29. Every exon of immuoglobulin gene corresponds exactly
with a know functional domain of the protein

31. Chromosomes, nucleosomes and controlling chromatin sturcture
How is DNA packed in the chromosomes.
Unusual chromosome structures.
Nucleosomes.
Controlling chromatin structure.
Gene function and chromatin structure.
Epigenetics (read Chapter 31 of Genes IX) .

32. DNA Topology
Topology is a branch of mathematics that studies the properties of an object that do not change under continuous deformations. For circular DNA molecules, a topological property is one that is unaffected by deformations of the DNA strands as long as no breaks are introduced. DNA topology is the study of the spatial relationship of DNA.
Topology of DNA
Intramolecular properties – relationship between the two strands of the duplex. Only CCC DNA or linear duplex with at least two anchors are of topological concerns.
Intermolecular properties – relationship between two molecules, eg., catenanes.

33. Supercoils

34. Supercoiling of DNA can only occur in closed-circular DNA or linear DNA where the ends are fixed.
Underwinding produces negative supercoils, whereas overwinding produces positive supercoils.

35. Negative and positive supercoils .
Topoisomerases catalyze changes in the linking number of DNA.

36. Relaxed and supercoiled plasmid DNAs

37. Topology of cccDNA is defined by: Lk = Tw + Wr, where Lk is the linking number, Tw is twist and Wr is writhe.

38. Intertwining of the two strands
Nodes = ss crossing on 2D projection.
Right-handed crossing = +1/2
Left-handed crossing = -1/2
Lk = number of times one strand winds around the
other on 2D projection.
One linking number = 2 nodes.

40. DNA Compaction Requires Solenoidal Supercoiling, not plectonemic supercoiling.

41. Chromosome, chromatin, chromatid
Chromatin: the complex of DNA and protein in the nucleus of the interphase cell. Heterochromatin refers to regions of the genome that are permanently in a highly condensed condition, while euchromatin refers to the rest of the genome.
Chromosome: consists of one DNA molecule and proteins. Visible as morphological entity only during mitosis.
Chromatids: copies of a chromosome produced by replication.

42. Packaging of DNA
Packing ratio: the length of the DNA divided by the length of the
unit that contains it.

45. Bacterial chromosome

46. HU and H1 proteins may be
involved in condensing DNA.
There are about 400 domains
of independent supercoiling,
each consists of ~10-40 kb.
The average density of
supercoiling is ~1 turn/100 bp.
4. Treatment with reagents that
act on RNA or protein may
unfold the nucleoid.
Question: How is #2-3 determined?

50. Loops, domains and scaffolds in eukaryotic DNA
Genome, when isolated carefully, can be visualized as 10 nm fiber, consisting of DNA and protein. Supercoiling measured by EtBr indicates about 1 negative supercoils per 200 bp.
Loops can be seen directly when the majority of histones are removed (see next Fig.). Threads of DNA emanate from the scaffold as loops of average length mm (30-90 kb).

52. Is DNA attached to the nuclear
matrix or scaffold via specific
sequences? Analysis of MAR
(Matrix attachment region) does
not reveal any conservation of
sequence in MAR fragments.
cis-acting sites that regulate
transcription are common. A
recognition site for
topoII is usually present in the
MAR.

55. Lampbrush chromosome

57. Polytene chromosomes
The length is the chromosome set is ~2000 mm, while
the DNA in extended form would stretch for ~40,000 mm,
so the packing ratio is about 20.

61. Centromeres:
The sequences required for centromeric function has been
identified in yeast S. cerevisiae (as shown below).
The centromeres of S. pombe lie within regions of
kb that consist largely of repetitious DNA. The primary motif
of primate centromeres is the a satelllite.

64. Telomeres: consists of repetitive sequences rich in G.

65. SS G-tails in Telomere
Incomplete replication of lagging-strand at linear DNA ends.

68. Minimum features required for existence as a chromosome (YAC)
Telomere to protect chromosome ends and to ensure survival.
A centromere to support segregation.
An origin to initiate replication.

69. How is DNA packed in the chromosomes
DNA supercoiling.
Proteins assisted packaging (nucleosomes)

71. The nuclosome is the subunit of all chromatin

72. Packing ratio ~6.

80. DNA structure varies on the nucleosomal surface

83. The average periodicity of DNA in nucleosome is 10.2-10.4
bp, which is slightly less than the 10.5 bp in free DNA.

84. Supercoiling and the periodicity of DNA: linking number paradox
SV40 DNA is 5.2 kb
(1500 nm). In virion
or infected nucleus, it
is packaged into a series
of nucleosomes with the
contour length of 210
nm. The no. of supercoils
measured approximates
the no. of nucleosomes.

85. Path of nucleosomes in the chromatin fiber

87. Packing ratio ~40, suggesting ~6 nuclosomes
for every turn.

89. Model of DNA compaction in eukaryotic chromosomes

90. Organization of the histone octamer

95. Reproduction of chromatin requires assembly of nucleosomes

96. Histone octamers are not conserved during replication,
Bit H2A-H2B dimers and H3-H4 tetramers are conserved.

97. Do nucleosomes lie at specific positions?

100. Position of DNA on nucleosome
can be important in controlling
access to DNA. Displacement of
the DNA by 10 bp changes the
sequences in the linker regions,
while a non-10 bp displacement
can affect accessibility of DNA
to different factors.

101. Are transcribed genes organized in nucleosomes?

104. The unifying model suppose that
RNA polymerase displaces histone
octamers as it progresses. If the
DNA behind the polymerase is
available, the octamer reattches
there. If the DNA is not available,
eg., another polymerase continues
immediately behind the first, then
the DNA may persist in an
extended form.

105. DNAase hypersensitive sites: usually found only in
chromatin of cells in which the associated gene is being
transcribed.

107. DNAase I sensitivity defines a
chromosomal domain, a region
of altered structure including at
least one active transcription unit.

109. 29.14 Insulators Block the Actions of Enhancers and Heterochromatin
Insulators are able to block passage of any activating or inactivating effects from:
Enhancers
Silencers
LCRs
Figure 29.42

110. Insulators may provide barriers against the spread of heterochromatin.
Figure 29.43

111. 29.15 Insulators Can Define a Domain
Insulators are specialized chromatin structures that have hypersensitive sites.
Two insulators can protect the region between them from all external effects.
Figure 29.44

112. 29.20 An LCR May Control a Domain
An LCR (locus control region):
is located at the 5′ end of the domain
consists of several hypersensitive sites
Figure 29.54

113. 29.21 What Constitutes a Regulatory Domain?
A domain may have:
an insulator
an LCR
a matrix attachment site
transcription unit(s)
Figure 29.54

114. Controlling Chromatin Structure

115. 30.2 Chromatin Can Have Alternative States
Chromatin structure:
is stable
cannot be changed by altering the equilibrium of transcription factors and histones
Figure 30.2

116. 30.3 Chromatin Remodeling Is an Active Process
There are several chromatin remodeling complexes that use energy provided by hydrolysis of ATP.
Figure 30.3

117. The SWI/SNF, RSC, and NURF complexes all:
are very large
they share some common subunits
A remodeling complex does not itself have specificity for any particular target site.
It must be recruited by a component of the transcription apparatus.
Figure 30.5

118. 30.4 Nucleosome Organization May Be Changed at the Promoter
Remodeling complexes are recruited to promoters by sequence-specific activators.
The factor may be released once the remodeling complex has bound.
Figure 30.6

119. The MMTV promoter requires a change in rotational positioning of a nucleosome to allow an activator to bind to DNA on the nucleosome.
Figure 30.7

120. 30.5 Histone Modification Is a Key Event
Histones are modified by:
methylation
acetylation
phosphorylation
Figure 30.8

121. 30.6 Histone Acetylation Occurs in Two Circumstances
Histone acetylation occurs transiently at replication.
Figure 30.12

122. Histone acetylation is associated with activation of gene expression.
Figure 30.13

123. 30.7 Acetylases Are Associated with Activators
Deacetylated chromatin may have a more condensed structure.
Transcription activators are associated with histone acetylase activities in large complexes.
Figure 30.14

124. Histone acetylases vary in their target specificity.
Acetylation could affect transcription in a quantitative or qualitative way.

125. 30.8 Deacetylases Are Associated with Repressors
Deacetylation is associated with repression of gene activity.
Deacetylases are present in complexes with repressor activity.
Figure 30.16

126. 30.9 Methylation of Histones and DNA Is Connected
Methylation of both DNA and histones is a feature of inactive chromatin.
The two types of methylation event may be connected.

127. 30.10 Chromatin States Are Interconverted by Modification
Acetylation of histones is associated with gene activation.
Methylation of DNA and of histones is associated with heterochromatin.
Figure 30.17

128. 30.11 Promoter Activation Involves an Ordered Series of Events
The remodeling complex may recruit the acetylating complex.
Acetylation of histones may be the event that maintains the complex in the activated state.
Figure 30.18

129. 30.12 Histone Phosphorylation Affects Chromatin Structure
At least two histones are targets for phosphorylation, possibly with opposing effects.

130. 30.13 Some Common Motifs Are Found in Proteins That Modify Chromatin
The chromo domain is found in several chromatin proteins that have either activating or repressing effects on gene expression.
The SET domain is part of the catalytic site of protein methyltransferases.
The bromo domain:
is found in a variety of proteins that interact with chromatin
is used to recognize acetylated sites on histones.