Anda di halaman 1dari 11

BIOCHEMISTRY

LECTURE # 3.01b: Nucleosides and Nucleotides, Nucleic Acid Structure and Function, DNA Organization
DATE OF LECTURE: January 18, 2018
INSTRUCTOR: Group 4, 2021B

OUTLINE
III. DNA Organization
A. Overview
B. Biomedical Importance of DNA Organization
C. DNA Organized Composition
D. Chromatin
E. Chromosomes
F. Mammalian Genome Function
G. Mitochondrial Cellular DNA
Figure 1. Vertical gene transfer.
H. Gene Alteration & Rearrangement
• Has a frequency to 1 in 106 cell divisions
III. DNA ORGANIZATION • Factors that increase rate of mutation:
o Viruses
A. OVERVIEW o Chemicals
o Ultraviolet and Ionizing radiation
• Reading Frame- a sequence of nucleotide triplets potentially
• There are several types of mutations (Table 1; Figures 2 &3)
translatable into an AA of Polypeptide; relies on initiating codon
o Often read Downstream (5’3’)
TABLE 1. DIFFERENT TYPES OF MUTATIONS
o Uumpisahan natin dito… ang translation.
MUTATION REMARKS
• Reading Phase- sequence of nucleotide triplets potentially
SINGLE BASE MUTATIONS (AKA POINT MUTATIONS)
processed by ribosome, RELATIVE to the Reading Frame.
o Clear? VERY GOOD. Base Substitution A nucleotide is replaced with another
Transversion Purine to pyrimidine (pyr) and vice versa
(Fig 2)
B. BIOMEDICAL IMPORTANCE Transition Purine to other purine; pyr to other pyr
• Genetic information may be transmitted or exchanged by these (Fig 2)
processes: TYPES OF POINT MUTATION BY EFFECT
• At 3rd base of codon (Wobble base)(1)
o Exact Replication- recall, information is copied from the Template Silent • No changes in amino acid (AA) of peptide
or Noncoding Strand
▪ Noncoding, since iba (complementary) yung sequence niya • Different AA is incorporated somewhere along the
Missense
sa mRNA, which then codes for protein peptide. May have the ff effects:
▪ For replication, you do NOT make mRNA; rather, you make Acceptable Replacement yields AA with similar properties (eg
a copy complementary to template strand (Consevative) Nonpolar branched) to original AA(2)
▪ Recall as well, 5’-cap is needed, NOT free or 5’-OH Replacement leads to a protein with partial but
Partially Acceptable
o Crossing over (aka Recombination) abnormal function
o Transposition Unacceptable aka
o Conversion Resulting protein is dysfunctional.
(nonconservative)
• Processes ensure adaptability (Multidrug-resistant TB anyone?) and Results to a Stop Codon (Table 2) and yields only to
diversity for the organism Nonsense
a fragment of intended protein molecule
o If processes are erratic, however, these result in disease MUTATION OF “MULTIPLE” BASES May alter Reading Frame
• There are numerous enzymes participating in DNA replication, • From deletion or insertion of NTs in DNA
alteration, and repair Frameshift (Fig 3)
• Affects mRNA, hence affecting Protein
Suppressor • Result of abnormal tRNA themselves
MUTATIONS 1Recall: Genetic Code (Table 2)- every codon (3 consecutive bases) codes for a
• Mutations- via change in base sequence of DNA; may result to: specific amino acid. Several codes varying mainly at the Wobble Base (3rd Base
o Faulty Replication, Movement, and/or Repair of Codon) encode for ONLY ONE AA
o Abnormalities in gene product (RNA, Protein function, or amount) 2Resulting protein is almost indistinguishable from normal protein
▪ Due to mutations in transcribed protein-coding and
noncoding DNA
o Vertical transmission- transmitted mutation in germ cell to an
offspring (Fig 1)
o Horizontal Transmission- transmission of effects of mutation on
somatic cells to successive generations, within an organism
o Several diseases, including Cancer, may arise from combined
effects of vertical and horizontal transmission of induced Figure 2. Diagrammatic representation of transitions and transversions.
mutations

Trans By: STH-川端, Landicho, Mortos Page 1 of 11


BIOCHEMISTRY
LECTURE # 3.01b: Nucleosides and Nucleotides, Nucleic Acid Structure and Function, DNA Organization
DATE OF LECTURE: January 18, 2018
INSTRUCTOR: Group 4, 2021B

CASE 2: DELETION OR INSERTION IS WITHIN NORMAL STOP OR


TERMINATION CODON
• Ah! GG Boi!
• May natanggal, or may nasiksik (Fig 3).
o Pansinin niyo sa normal, kung anu-ano ang “CODONS”- eg DOG, HIT,
THE, etc.
o CAUTION: If one or two NTs are removed, there WILL be alteration of
the reading frame and reading phase
o Tignan niyo, nung nadelete si D, nag-iba na yung codon, from DOG,
naging OGH. Nausog ang reading frame.
o So, lahat ng susunod na bases distal to the alteration ay altered
o Hence, Sobrang Iba ang protein product. Two cases:
▪ Severely deviated polypeptide (PETMALU)
➢ May also be LONGER; if mutation is before stop codon,
Figure 3. Different types of mutations illustrated. Deletion and Insertion MAY mag-iiba na rin ang reading frame
both lead to Frameshift Mutations. ➢ Tutuloy ang translation until the next stop codon
▪ Prematurely terminated polypeptide (Putol, Bitin, Naiwan sa ere,
Paasa, Nawalan ng gana ang tadhana, etc.)
TABLE 2. GENETIC CODE (CODON ASSIGNMENTS IN MAMMALIAN
o NOTE: If three nucleotides are deleted, as in Fig 3, there is no
mRNAs)
alteration of the reading phase
SECOND NUCLEOTIDE (NT) THIRD NT
FIRST NT ▪ Protein product is ALMOST similar, EXCEPT that it is missing
U C A G
corresponding number of amino acids
Phe Ser Tyr Cys U
• If insertion occurs upon deletion (vice versa), reading frame may be
Phe Ser Tyr Cys C reestablished.
U
Leu Ser Stop(2) Stop(2) A o AAs are altered between insertion and deletion sites.
Leu Ser Stop(2) Trp G o Beyond these sites, AA sequence is correct
Leu Pro His Arg U o Protein products (with abnormal parts, surrounded by normal
Leu Pro His Arg C sequences) are linked to certain diseases
C
Leu Pro Gln Arg A
Leu Pro Gln Arg G
Ile Thr Asn Ser U
Ile Thr Asn Ser C
A
Ile Thr Lys Arg A
Met(1) Thr Lys Arg G
Val Ala Asp Gly U
Val Ala Asp Gly C
G
Val Ala Glu Gly A
Val Ala Glu Gly G
*Recall: mRNA is the one translated to protein, hence, the respective bases
in Table 2
Tandaan- First, Second, and Third nucleotides refer to individual NTs of a
codon, read from 5’3’ (DOWNSTREAM), left to right
1You may have been taught, AUG is the Start Codon. In these cases, ang

First Residue sa Initial Protein Product is Methionine.


o Met disappears via Post-translational Modifications
Figure 4. The different types of Missense mutations.
Mahaba-habang usapan ‘to. Consider NORMAL tRNA function. Ganito yan:
• Missense Mutations
o Acceptable Missense: In Fig 4, Hb Hikari has normal physiologic
CASE 1: DELETION OR INSERTION IS DISTAL TO NORMAL
properties but is altered electrophoretically.
TERMINATION OR STOP CODON (LAGPAS SA STOP CODON)
o Partially acceptable Missense: Hb S only has partial function; can bind
• Ah! No Problem. Di naman magtatranslate sa protein since translation O2 but precipitates when deoxygenated
has stopped prior to the reading of mutation. ▪ Cases RBCs to sickle
▪ Sickle Cell Disease Molecular Basis
o Unacceptable Missense: Fe2+ oxidation to Fe3+; will NOT bind Oxygen

Trans By: STH-川端, Landicho, Mortos Page 2 of 11


BIOCHEMISTRY
LECTURE # 3.01b: Nucleosides and Nucleotides, Nucleic Acid Structure and Function, DNA Organization
DATE OF LECTURE: January 18, 2018
INSTRUCTOR: Group 4, 2021B

SUPRESSOR MUTATIONS HISTONES


• Occurs in PROKARYOTIC and lower eukaryotic organisms • The MOST ABUNDANT CHROMATIN proteins
• Some of Suppressor tRNA can decode altered codons • Small family of closely-related proteins (Table 3)
o Suppresses effects of mutations in distinct mutated mRNA- o Carboxyl terminal two-thirds are Hydrophobic
encoding structural genes o Amino terminal thirds are BASIC
o HOWEVER, abnormal tRNA can’t distinguish from normal and ▪ RECALL: Ensures good attraction & interaction with DNA via
altered codons negatively-charged DNA backbone.
▪ Leads to less-viable cells ▪ Amino terminals are said to extend, available for PTMs
• Suppressor tRNA may be used to incorporate unnatural AAs at defined
sites (USEFUL in RESEARCH) TABLE 3. DIFFERENT TYPES OF HISTONES
HISTONES REMARKS
OTHERS • LEAST-Tightly bound to chromatin.
• Telomere Shortening (elaborated in section E)- associated with aging • Stabilizes 30nm Chromatin Fiber
H1
and malignant transformation (cancer) • EASILY removed by salt solutions(1)
o When removed, yields Nucleosome
MAJOR HISTONES IN NUCLEOSOME (aka CORE HISTONES)
C. COMPOSITION OF DNA
(Structures tightly conserved between species)(2) (Fig 6)
Kindly see Section II. A & C of Trans 3.01a for a review. However, H2A Forms dimers (H2A-H2B).
you should know the composition of DNA by now. H2B Firmly binds 2 additional half turns (0.5+0.5=1 turn)
H3 Forms tetramers with 2 molecules each (H3-H4)2
H4 Has central role in nucleosome; binds 0.75 turns
D. CHROMATIN 1TAKE NOTE: This is very useful in DNA purification techniques. There is this

washing step with saline solution when purifying DNA. This is the rationale.
OVERVIEW 2The mere fact na may conservation among species implies na pare-parehas

• Chromatin- the chromosomal material in nuclei of cells of eukaryotic function nila in ALL eukaryotes and role is specific for these.
organisms. Consists of:
o Very long, double-stranded DNA (dsDNA) • Core Histones- form nucleosomes
o Histones & Non-Histone Proteins o Histone oligomers form Histone Octamer under physiological
o Small amount of RNA conditions (H3-H4)2-(H2A-H2B)2.
• Histones- nearly similar in mass with the DNA o Subject to at least six types of covalent or Posttranslational
o Basic in nature. Why? Basic proteins (with Basic AAs eg Lys, His, Modification (PTM):
or Arg) have net Positive Charges ▪ Acetylation
o Meanwhile, DNA has Net Negative Charges due to Phosphate ▪ Methylation
Backbone. Anong mangyayari? Ionic attraction(Buti pa sila, ‘no?) ▪ Phosphorylation
• Non-histone Proteins- acidic and larger than histone proteins ▪ ADP-Ribosylation
o Includes enzymes involved in DNA replication and repair; proteins ▪ Monoubiquitylation
for RNA synthesis, & transport to cytoplasm ▪ SUMOylation (SUMO=Small, Ubiquitin-related Modifier)
• Nucleosome- complex of certain Histones and DNA (Fig 5) o These enable histones to perform various roles (Table 4)

TABLE 4. MODIFICATION & ASSOCIATED ROLES OF HISTONES


MODIFICATION ASSOCIATED PROCESS REMARKS
(In)/activation of gene
H3 and H4 Acetylation 
transcription
General core histone
Chromosome assembly During Replication
Acetylation
Activation and repression of
Histone Methylation 
gene transcription
H1 Phosphorylation Chromosome condensation During Replication
ADP-Ribosylation of
DNA Repair
Histones
; Heterochromatic
Monoubiquitylation Gene Regulation
Gene silencing
SUMOylation Transcription -
()- Activation and/or Repression (Inactivation); (-)- Inactivation
Figure 5. ECM of Nucleosomes (White Balls “on String”). These are also
referred to as “Rosary Beads”. Tignan niyo itsura oh.

Trans By: STH-川端, Landicho, Mortos Page 3 of 11


BIOCHEMISTRY
LECTURE # 3.01b: Nucleosides and Nucleotides, Nucleic Acid Structure and Function, DNA Organization
DATE OF LECTURE: January 18, 2018
INSTRUCTOR: Group 4, 2021B

NUCLEOSOMES • 30nm Chromatin Fiber


• Contains Histones & DNA; “Bead” Structure (Fig 5), linked by DNA o Nucleosome supercoiled 6-7 times (yung Beads-on-string, inikot mo pa,
• Same in structure, whether freshly isolated, or when Histone Octamer 6-7 times).
is mixed with purified dsDNA, under physiological conditions. o Stabilized by H1 Histones
o Lead to elucidation of structure in Figure 6. o Forms Mitotic Chromosome when folded 100-Fold
▪ Histone octamer- composed of two of each of Core o Forms Interphase Chromosomes when further organized into 30000-
Histones 100000bp loops or domains attached to Scaffolding
▪ 145bp (1.75 helical turns) interact with Histone Octamer ▪ Scaffolding is aka Nuclear Matrix
▪ Note how Histone H1 is weakly associated. o Among these domains, some DNA sequences are nonrandom
o Certain genes are mobile,moving obligatorily to certain loci in nucleus;
aka Jumping Genes
o Folded into Looped Domains in metaphase chromosomes
▪ Anchored to nuclear matrix by Lamins- proteins constituting
integral components of inner nuclear membrane in nucleus

Figure 6. Nucleosome structural model.

• Nucleosomes may be reconstituted, regardless of organism of origin,


H1 or non-histone proteins.
o Iwan mo lang ang solution ng pure dsDNA & Core histones,
mabubuo at mabubuo ang Nucleosome core.
• DNA is supercoiled via left-handed helix over the surface of disk-
shaped histone octamer
o Recall, 0.75 turn (H3-H4)+0.5 +0.5 turns (H2A-H2B)=1.75 turns
▪ 2 H2A-H2B dimers are added to stabilize the complex.
▪ 1.75 superhelical turns of DNA are wrapped around surface
of histone octamer. (145-150bp)
• Nucleosome core particles are linked by ~30bp region of DNA, aka
LINKER
• All these structures repeat, giving a “Rosary”-like or “Beads-on-string”
like appearance when analyzed by electron microscopy.

• Histone Chaperones-proteins with high-affinity histone binding


o Facilitates Chromatin Assembly Factors (mediators of
nucleosome assembly)
• As nucleosomes are assembled, histones are released from respective
histone chaperones
• Phasing- nonrandom distribution of regions on specific DNA
molecules preferred by nucleosomes, related to:
o Physical flexibility of nucleotide sequences for kinks in supercoils
o DNA-Bound factors (limit sites of nucleosome deposition)

HIGHER-ORDER STRUCTURES FOR COMPACTION OF CHROMATIN


Two Higher-Order Structures:
• 10nm Fibril (aka Beads-On-String)
o Nucleosome disks (w/ 10nm diameter & 5nm height) separated by
30bp Linkers parallel to Fibril Axis
Figure 7. Higher-Order Structures in Chromatin (Two structures above DNA)
• Pansinin niyo, nung inikot sa histones yung DNA, mas naging compact siya.
So what more nung inikot siya further into 30nm Chromatin fibers?
o Otherwise, sobrang sabog ng DNA natin, right?

Trans By: STH-川端, Landicho, Mortos Page 4 of 11


BIOCHEMISTRY
LECTURE # 3.01b: Nucleosides and Nucleotides, Nucleic Acid Structure and Function, DNA Organization
DATE OF LECTURE: January 18, 2018
INSTRUCTOR: Group 4, 2021B

‘ACTIVE’ VS ‘INACTIVE’ CHROMATIN • Rich in meC (5-methyldeoxyCytidine)


• Every cell of individual metazoan (eukaryotic, non-fungi) organism • Histones in this region has higher “repressing” modifications vs “activating”
contains same genetic information covalent modifications
o Eg. Gastric chief cells express gene for Pepsinogen, while skin • One of the members of mammalian female’s X Chromosomes are
cells do not. BUT both genes are present in these cells. heterochromatic. However, this Heterochromatic X Chromosome
o Differences between cell types are explained by levels of o May decondense during gametogenesis
expression of common genetic information o May become transcriptionally active during early embryogenesis
▪ This property is exploited in DNA Microarrays (Fig 8) • Has two types: Constitutive and Facultative (Table 5)
▪ Eg Genes for proliferation may be expressed in higher
amounts in cancer cells, as compared to normal cells. TABLE 5. CONSTITUTIVE VS FACULTATIVE HETEROCHROMATIN
PROPERTY CONSTITUTIVE FACULTATIVE
Condensation + 
Activity - 
Near chromosomal centromere;
Location
Chromosomal ends (Telomere)
(+) -present/observed (-)-not present/observed ()- present/not present

(Refer to Table 5)
Constitutive Heterochromatin
Figure 8. DNA Microarray color legends (left) and actual DNA Microarray. • Always condensed, therefore, essentially inactive
Black color= neither normal nor cancer cells express gene. o Recall, if chromatin is condensed, access by transcription enzymes are
limited. Makes sense? Good.
ACTIVE REGIONS Facultative Heterochromatin
• Active regions contain altered nucleosome structures • May be condensed or uncondensed (hence may appear as Euchromatin)
• DNA in these regions contain long (~100000bp) regions more o Hence, may be inactive or actively transcribed
susceptible to DNase I digestion o Example: Heterochromatic X Chromosome of Mammalian females
o DNase I- makes single (1) stranded cuts in any DNA segment
(low sequence specificity)
▪ DNase I digests DNA unprotected or unbound by proteins E. CHROMOSOMES
into component deoxynucleotides
o Sensitivity of active chromatin to DNase I reflects transcription OVERVIEW
potential only, not transcription itself. • Chromosomes-eto na yung alam nating X-shaped rods (Figure 9)
▪ Sensitivity may rather be correlated to lack of 5- o Formed by further organization of chromatin and chromatid (Fig 7)
methyldeoxycytidine (meC) in DNA and specific histones • Has twofold symmetry at metaphase
variants or Histone PTMs (Recall Table 4) • Centromeres- adenine-thymine (A-T)- rich region containing repetitive
• DNA in active regions also have shorter regions (100-300nt) sequences ranging from 102 (yeast) to 106 (human) base pairs
hypersensitive to DNase I (10-fold more sensitive to DNase I) o Site where identical sister chromatids are connected
o May be due to favorable access of nuclease to DNA o Its relative position is characteristic for each type of chromosome
o Located upstream (5’) of active gene o Bound by H3 Histone variant, CENP-A & other Centromere-Binding
o Site of Enhancers (DNA region favorably binding transcriptional Proteins
activator proteins) o Kinetochore- centromere-protein complex
o Marker for transcription control elements ▪ Provides anchor for mitotic spindle
• Stains lightly under microscope (aka Euchromatin. Recall Histo) ▪ Essential for chromosomal segregation in mitosis
• In certain insects (eg Chironomus & Drosophilia), active regions of
their Polytene Chromosomes are decondensed into ‘Puffs’
o Sites of RNA synthesis; contains transcription enzymes
o Useful in gene mapping
▪ In humans, these Polytene chromosomes need not be
formed for gene mapping c/o highly specific FISH
(Fluorescence In-Situ Hybridization) techniques
➢ From the name itself, Hybridization using fluorescent-
labeled NTs (probes)

INACTIVE REGIONS
• Densely packed in Interphase, aka Heterochromatin
o Stains heavily under microscope. Recall Histo.
o Packing does not provide access to DNase I and to transcription
enzymes. Makes sense? Figure 9. Sister chromatids of mitotic Human Chromosome 12

Trans By: STH-川端, Landicho, Mortos Page 5 of 11


BIOCHEMISTRY
LECTURE # 3.01b: Nucleosides and Nucleotides, Nucleic Acid Structure and Function, DNA Organization
DATE OF LECTURE: January 18, 2018
INSTRUCTOR: Group 4, 2021B

• Telomeres- ends of each chromosome • Recall that chromosomes, when unpacked, will give you dsDNA.
o Made of short T-G repeats o As seen in Fig 10, from the uncondensed material in interphase, arise
o Have repeats of sequence 5’-TTAGGG-3’ the condensed chromosomal structures in metaphase.
o Telomerases- for telomere synthesis and maintaining telomere o As DNA is further organized, its packing or compaction ratio
length increases
▪ Multisubunit RNA template-containing complex related to o Packing ratios of each order of DNA structures (Fig 7), up to the
viral RNA-dependent DNA Polymerase (reverse chromosomes are summarized in Table 6.
transcriptase) • Packaging of nucleoproteins within chromatids is not random, as observed in
▪ Target in Cancer chemotherapy & drug development Quinarine or Giemsa staining (Fig 11)
▪ For synthesis of telomere and maintenance of its length o Within members of the same species, staining patterns are
• Each sister chromatid contains one dsDNA molecule reproducible
o Contrary to heterochromatin, dsDNA is less densely packed in o However, staining patterns are different, between species (even
interphase as compared to condensed chromosome in metaphase between related species)
o Metaphase chromosomes are nearly transcriptionally inactive ▪ Suggests Species-Specific characteristics of DNA
o Human haploid genome contains 3x109bp & 1.7x107nucleosomes,
hence 23 chromosomes has average of 1.3x108NTs in dsDNA CODING & NONCODING REGIONS
▪ Eto yung reason for the need of 8000-fold compression ng
dsDNA, just to form metaphase chromosome (Fig 10)

Figure 10. Comparison between interphase and metaphase cells. Note the
difference in structures of nuclear material in each stage.

PACKAGING
Figure 12. Relationship between chromosomal DNA & mRNA.
TABLE 6. PACKING RATIO OF EACH ORDERS OF DNA STRUCTURE
CHROMATIN FORM PACKING/ COMPACTION RATIO • Protein-coding regions of DNA are interrupted by large intervening
Naked double-helical DNA ~1.0 sequences of nonprotein-coding DNA
10nm Fibril of nucleosomes 7-10 o Recall: Protein-coding regions- lead to single mRNA in cytoplasm
30nm Chromatin Fibers (in o Primary transcripts of DNA, mRNA precursors, contain noncoding
50-60
superhelical nucleosomes) intervening sequences of RNA (Fig 12)
Condensed Metaphase ▪ These transcripts were initially termed hnRNA, heterogenous
8000
Chromosome loops (size and lengthwise) nuclear (nucleus-restricted) RNA
▪ Intervening sequences must be removed in a process wherein
appropriate coding sequences are also joined (Splicing) (Fig 12,
primary mRNA transcript  mRNA)
▪ Introns- intervening, nonprotein-coding sequences longer than
exons (green regions, Fig 12)
▪ Exons- actual protein-coding sequences, shorter than introns
(blue regions, Fig 12)
▪ Eukaryotic genes have alternating introns and exons
o Refer again to Fig 12. Genes possess a regulatory region (red-
hatched area) at the 5’-end and may or may not be adjacent to
transcription initiation site (bending arrow)
o Precursor mRNA can undergo alternate splicing
▪ Yields increased number of distinct (but related) proteins just from
a single gene and corresponding transcript
o Function of introns are not completely defined
▪ Introns separate exons, such that genetic rearrangements
(recombination) are enabled rapidly
Figure 11. Human karyotype of a male (note the XY Chromosome) of
➢ Allows rapid evolution of biological function
metaphase chromosomes.

Trans By: STH-川端, Landicho, Mortos Page 6 of 11


BIOCHEMISTRY
LECTURE # 3.01b: Nucleosides and Nucleotides, Nucleic Acid Structure and Function, DNA Organization
DATE OF LECTURE: January 18, 2018
INSTRUCTOR: Group 4, 2021B

o Intronic DNA of certain genes may other contain coding and ✓ 500,000 copies in genome
noncoding RNAs ✓ ~10% of human genome
✓ Transcribed as integral components of mRNA
precursors or as discrete RNA molecules
F. MAMMALIAN GENOME FUNCTION ✓ Includes 4.5S and 7S RNA, members highly
• Exact function of much of the mammalian genome is not well- conserved within a species & between
understood. mammalian species
• Haploid genome of each human cell consists of 3x109bp of DNA ✓ Alu B1 & Alu B2 RNAs - regulators of mRNA
subdivided into 23 chromosomes production at levels of transcription/splicing
o ~1% is exonic DNA ✓ Possibly mobile elements, due to structural
▪ This exonic DNA was shown through mutation studies & similarities with retroviral terminus.
genome complexity to code for only <100,000 proteins ❖ May be mobile elements (transposons)
o Current estimates suggest <25,000 protein-coding genes in ✓ CLINICAL SIG.: Neurofibromatosis
humans. Parang ang konti, no? Konti talaga yan! ✓ As result of a disastrous transposition event
▪ Implies most of genome is noncoding
▪ Of course, some are used as regulatory regions (regulates ➢ Both are retroposons- originated from transposition or
gene expression) transfer from one location to another
▪ Some excess- introns ❖ Via RNA intermediate by Reverse Transcriptase
▪ Others- families of repetitive sequences with undefined (enzyme transcribing RNA template to DNA)
functions
▪ ENCODE Project Consortium has shown that a large o Highly Repetitive
fraction of transcriptions generate lncRNA (Recall Trans ▪ Present in 1 to 10,000,000 copies per haploid genome
3.01a, Table 7, section II. F) ▪ 5-500bp repeated many times in tandem
• DNA in eukaryotic genome can be subdivided into unique ▪ Clustered in centromeres and telomeres of chromosome
(nonrepetitive)- and repetitive-sequence DNA ▪ Majority are transcriptionally inactive
▪ Some play structural role in chromosome
UNIQUE (NONREPETITIVE)- DNA
• Unique-sequence DNA includes single copy, coding genes. o Microsatellite (MS) Repeat Sequences
• >50% of DNA in eukaryotes ▪ Both dispersed & grouped tandem arrays
▪ 2 to 6bp repeated up to 50 times
• Though coding for proteins, not all of these genes are required for
▪ Act as gene markers; marks location of gene in chromosome
viability. Eg Saccharomyces cerevisiae needs only 1/5 of its coding
▪ Useful in creating gene linkage maps
genes to live
▪ Heritable trait; detected by Polymerase Chain Reaction
• Recall: each of these genes are expressed in varying degrees in
➢ Polymerase Chain Reaction (PCR)- Process for making
different tissues
multiple copies of DNA
o Eg, skin is not involved in digestion, hence doesn’t express
➢ Using this technique, large number of family members can
Pepsinogen, though skin cells have gene for pepsinogen
be screened for MS Polymorphism
❖ Polymorphism may be associated in affected members
REPETITIVE-SEQUENCE DNA
while unassociated in unaffected
• Repetitive-sequence DNA has from 2 to 107 copies per cell. ❖ This is the first clue of genetic basis of disease
• 30% of Genome
• Two classes: ▪ Most commonly found as dinucleotide AC-repeats on one strand
o Moderately Repetitive and TG on opposite strand
▪ Present in <106 copies per haploid genome ➢ AC repeats occur at 50,000-100,000 genome locations
▪ NOT clustered; instead, interspersed with unique ➢ Other forms include CG, AT, & CA repeats
sequences
▪ In many cases, transcribed by RNA Polymerase II, with ▪ Number of copies-heterozygous in humans c/o variability in
caps similar from those on mRNA numbers on two chromosomes
▪ Microsatellite Instability- due to trinucleotide repeats
▪ Further classified into two more classes based on length: ➢ Increase in numbers can cause diseases (Table 7)
➢ Long Interspersed repeat sequences (LINES) TABLE 7. DISEASES ASSOCIATED W/ TRINUCLEOTIDE REPEATS
❖ 6 to 7 kbp lengths TRINUCLEOTIDE DISEASE
❖ 20,000-50,000 copies in mammalian genomes Unstable (CGG)n FraGile X Syndrome
❖ Species-Specific family of repeat elements Huntington’s Chorea
CAG Spinobulbar Muscular Atrophy
➢ Short Interspersed repeat sequences (SINES)
Kennedy Disease
❖ 70-300bp lengths (WAY shorter vs LINES)
CTG MyoTonic dystrophy
❖ >100,000 copies in human genome
Guide: Notice 1st & 3rd sequences are C&G, resp. Just remember, CGG=fraGile,
❖ Alu Family
CTG=myoTonic, then the rest, CAG.

Trans By: STH-川端, Landicho, Mortos Page 7 of 11


BIOCHEMISTRY
LECTURE # 3.01b: Nucleosides and Nucleotides, Nucleic Acid Structure and Function, DNA Organization
DATE OF LECTURE: January 18, 2018
INSTRUCTOR: Group 4, 2021B

▪ In some cases, deletions in mtDNA occur during oogenesis,


G. MITOCHONDRIAL CELLULAR DNA hence, not inherited from the mother
• Comprises 1% of cellular DNA ▪ mtDNA mutationsmyopathies, neurologic disorders, certain
• 54 out of 67 mitochondrial polypeptides- encoded via nuclear genes forms of DM
o The rest are encoded by Mitochondrial DNA (mtDNA) genes
(Tables 8 & 9).
H. GENE ALTERATION AND REARRANGEMENT
TABLE 8. MAJOR FEATURES OF mtDNA • Mutation- results from alteration of genetic material (Recall Table 1)
FEATURE REMARKS • There are several ways alteration can occur (Table 10)
Circular, double stranded (one Heavy, H TABLE 8. PROCESSES LEADING TO GENE ALTERATION &
Structure
& one Light, L chain/strand) REARRANGEMENT
Number of Base Pairs 16,569bp PROCESS OCCURENCE REMARKS
13 protein subunits of respiratory chain • Exchange of genetic information between
(total of ~67) homologous chromosomes
• 7 subunits, NADH dehydrogenase • Requires alignment of homologous
(Complex I) (similar) metaphase chromosomes
• Cyt b of Complex III • Crossing-Over occurs between these
Encodes for (see Table 9)
• 3 subunits, COX (Complex IV) chromosomes (Fig 13)
• 2 subunits, ATP synthase o Results in equal & reciprocal
During
Large (16S) and small (12S) mt exchange of genetic material
Chromosomal Meiosis in
ribosomal RNAs o Occurs between Homologous
Recombination Mammalian
22 mt tRNA molecules (Similar) chromosomes
Cells
UGA (standard Stop) Trp • Unequal Crossovers (Fig 14)
Genetic Code deviations
AGA, AGG (standard Arg) Stop o Unequal exchange
Unstranslated sequences Very few o One chromosome gets more (aka
Mutation rate High (5-10 times that of nuclear DNA) Insertion or Duplication)
Comparisons provide evidence about evolutionary o The other, less (aka Deletion)
Others o Results in disorders (eg  Lepore)
origins of primates & other organisms
o Affect number of repeats/tandem array
TABLE 9. MAP OF mtDNA GENES • Bacteriophage (bacterial virus)-
PROTEIN FAMILY/ PROTEIN ENCODED recombine w/ host DNA via incorporation
RNA INVOLVED HEAVY STRAND LIGHT STRAND • Circular bacteriophage & host DNA are
RNA broken, then recombined (Fig 15)
Ribosomal RNA (rRNA) 16S & 12S mt rRNA • Two options with respect to Host DNA:
Thr, Ser, His, Arg, Gly, Lys, Pro, Glu, Ser, Tyr, o Bacteriophage has Homologous
Transfer RNA (tRNA) Chromosomal Occurs with
Asp, Trp, f-Met, Ile, Leu, Cys, Asn, Ala, Gln, sequence as host Homologous
for Amino Acid Integration some viruses
Val, Phe (PESCY NAQ) Chromosomal Recombination
Respiratory Chain Protein Subunits o Bacteriophage synthesize protein
NADH-Coenzyme ND1, ND2, ND3, binding it to Nonhomologous site
ND6 ▪ Site-Specific
Q oxidoreductase ND4/ND4L, ND5
Cytochromes (Cyt) Cyt b, COX1, COX2, COX3 • Integration may be direct/indirect (eg RNA
ATP Synthase virus, HIV, via Reverse Transcriptase)
ATPase 6, ATPase 8 • “Jumping Genes/DNA” (Eg Alu Family)
(ATPase)
OTHER FEATURES • Lead to Processed Genes
Origin of Endogenous (at o Eg Genes for Immunoglobulin, -
Terminal (at 16,569th bp) globin
Replication ~11,000th bp)
Promoter Two (PH1 & PH2) One (PL) o DNA sequence=mRNA (Anong mali?)
COX- Cytochrome c oxidase ▪ DNA has exact sequence, without
Guide: Mas kaunti ineencode ni Light strand, so kahit yun lang aralin Introns, and poly-A tail added
niyo. The rest, kay Heavy Strand na. Eukaryotic ▪ Hence, actual mRNA (without its
Transposition
Cells introns & w/ poly-A tail) was reverse-
• Human mitochondria contains 2-10 copies of Small, Circular (~16kbp) transcribed, then integrated into the
dsDNA molecule DNA via Transposition event
o All mitochondria are contributed by Ovum during zygote formation o Contains short, terminal repeats at
▪ Thus, mtDNA- inherited by maternal, non-Mendelian means each end, like those in lower organisms
▪ Diseases from mtDNA mutation affecting a mother are o Pseudogenes- processed genes
passed to ALL her children, but only daughters will transmit containing Nonsense Codons;
product of evolution

Trans By: STH-川端, Landicho, Mortos Page 8 of 11


BIOCHEMISTRY
LECTURE # 3.01b: Nucleosides and Nucleotides, Nucleic Acid Structure and Function, DNA Organization
DATE OF LECTURE: January 18, 2018
INSTRUCTOR: Group 4, 2021B

• Like sequences on chromosomes may


occasionally pair up
o Removes mismatched sequences
Gene Rapid between them
Conversion changes • Leads to accidental fixation of variants
throughout family of repeats
o Homogenizes sequences of
members of repetitive DNA families
• After S phase, cells have tetraploid DNA
(ie the usual x-shaped Chromosome)
Sister
Diploic • Crossing-over can occur between
Chromatids
Eukaryotes genetically-identical sister chromatids
Exchange
• Has no significant consequence if
crossover is equal
Figure 15. Viral DNA integration into host DNA. Note the consequent ordering
• During development & differentiation
of genes.
• Eg VL & CL genes encoding for
Immunoglobulin G (IgG) light chain
variable and conserve portions, resp. REFERENCES
o Though encodes for a Single IgG
Immunoglobulin Rodwell, V., Bender, D., Botham, K. M., Kennelly, P. J., & Weil, P. A.
molecule, these genes are separated
Mammalian (2015). Harpers Illustrated Biochemistry 30th Edition. McGraw Hill
Gene in germline. (Malayo sila sa isa’t-isa)
Cells Professional.
Rearrangement o In a differentiated IgG-producing cell,
these genes are brought closer within
a single transcription unit (rearranged) Natapos mo ba? Rest, then back to the fight.-J
o An intron is transcribed in between
these genes, removed, then genes are
spliced in processing the IgG mRNA.

Figure 13. The process of crossing-over between homologous


chromosomes, generating recombinant chromosomes.

Figure 14. Unequal Crossover, producing Lepore & Anti-Lepore


hemoglobins (ie -Lepore)

Trans By: STH-川端, Landicho, Mortos Page 9 of 11


BIOCHEMISTRY
LECTURE # 3.01b: Nucleosides and Nucleotides, Nucleic Acid Structure and Function, DNA Organization
DATE OF LECTURE: January 18, 2018
INSTRUCTOR: Group 4, 2021B

15. Which of the following are higher-order structures for chromatin compaction?
QUIZ A. 10nm Chromatin Fiber B. 30nm Chromatin Fiber
1. The following statements are false, EXCEPT C. 100nm Chromatin Fiber D. 30nm Fibril
A. The template strand has a similar sequence as mRNA
B. Template strand is also known as the Coding strand 16. Answer in 15 is stabilized by ________ & anchored to ________
C. Template strand is processed into mRNA A. H1 Histones, Lamins B. Lamins, H1 Histones
D. None of the above. C. H2A Histones, Lamins D.Chromatin Assembly Factors, Histone Chaperones

2. Which is the complementary strand of this template with this sequence: 3’- 17. The following is false, concerning Active Regions, EXCEPT
AATTGGCCAT-5’ A. All cells contain the same genetic information, and expresses them in a similar
A. 3’ TTCCGGTA 5’ B. 5’ TTAAGGCCTA 3’ manner
C. 5’ TTAACCGGTA 3’ D. 3’ AATTCCGGAT 5’ B. DNA is densely packed in the interphase
C. Contains a DNase I sensitive region
For nos 3-5. D. Rich in 5-methyldeoxycytidine
A. H1 B. H2A C. H2B D. H3 E. H4
3. Histones found in nucleosome core, EXCEPT 18. The following is true, concerning Inactive Regions, EXCEPT
4. Histone that can readily be washed by saline solution A. Rich in 5-methyldeoxycytidine
5. Forms oligomers, EXCEPT B. Chromatin is densely packed in the interphase
C. Site of enhancers
For nos 6-9 D. Aka Heterochromatins
A. General Core Histone Acetylation B. H1 Phosphorylation
C. ADP-Ribosylation of Histones D. SUMOylation 19. Which of the following is true about Facultative & Constitutive
Heterochromatin?
6. Chromosome condensation A. Constitutive chromatin is not condensed
7. Chromosome assembly B. Constitutive chromatin is active
8. DNA Repair C. Facultative chromatin is in the condensed form only
9. Transcription D. Constitutive chromatin is found at chromosomal ends

10. The following are regulators of gene expression, EXCEPT For nos 20-23
A. H3 & H4 Acetylation B. Histone Methylation A. Telomeres B. Kinetochore C. Centromeres D. Chromatids E.
C. ADP Ribosylation of Histones D. Monoubiquitylation Chromosomes

11. The following statements concerning Nucleosomes are true, EXCEPT 20. A-T- rich region
A. Nucleosomes are made up of Histones & DNA 21. Bound by H3 Histone variant
B. Nucleosomes are characterized as “Beads on Strings” 22. T-G rich region
C. Freshly-isolated nucleosomes are different from those made by mixing 23. 8000-fold compressed DNA structure
Histone octamers with purified double-stranded DNA under physiological
conditions 24. Which of the following concerning the Alu family is false?
D. Histone octamer is made up of two each of the core histones A. They are transcribed as integral components of mRNA precursors or as
discrete RNA molecules
12. Nucleosome core particles are connected by ~30bp region of DNA, B. They include 4.5S and 7S RNA
known as C. Alu B1 & Alu B2 RNAs are regulators of mRNA production
A. Histones B. Chaperones C. Assembly Factors D. Linkers D. Alu family is fixed within the genome

13. The following makes up Chromatins, EXCEPT 25. Sequence leading to Fragile X syndrome
A. Very long, double-stranded DNA (dsDNA) A. CTG B. CAG C. CGG
B. Histones & Non-histone proteins
C. RNA 26. The following are caused by the sequence, CAG, EXCEPT
D. Chromatids A. Huntington’s Chorea B. Spinobulbar Muscular Atrophy
C. Kennedy Disease D. Myotonic Dystrophy
14. Which of the following concerning Phasing is/are False?
A. Phasing is the nonrandom distribution of regions on specific DNA 27. The following are true for the light chain of mitochondrial DNA (mtDNA),
molecules preferred by nucleosomes EXCEPT
B. Physical flexibility of nucleotide sequence for kinks in supercoils can affect A. Has an endogenous origin of replication
phasing B. Encodes for 16S & 12S mt rRNA
C. DNA-Bound factors limit sites of nucleosome deposition C. Encodes for Proline
D. All of the above D. Encodes for ND6
E. None of the Above E. Has one promoter

Trans By: STH-川端, Landicho, Mortos Page 10 of 11


BIOCHEMISTRY
LECTURE # 3.01b: Nucleosides and Nucleotides, Nucleic Acid Structure and Function, DNA Organization
DATE OF LECTURE: January 18, 2018
INSTRUCTOR: Group 4, 2021B

28. The following are true, EXCEPT 6. B. Recall Fig 7. H1 is involved in Chromosome condensation
A. Chromosomal integration occurs in mammalian cells 7. A. Chromosome assembly requires core histones to be acetylated.
B. Chromosomal integration occurs with certain viruses 8. C. There are several DNA repair mechanisms. Eitherway, it may require
C. Transposition involves the Alu Family breaking up the Histone-DNA complex, to provide repair enzymes access.
D. Gene rearrangement homogenizes sequences of members of repetitive One plausible manner is via ADP-Ribosylation of Histones
DNA families. 9. D. Small Ubiquitin-related Modifier is done to a histone in transcription.
E. All above statements are true. 10. C. Recall, this is involved in DNA Repair. The rest are regulators of gene
expression.
29. The following are false, EXCEPT 11. C. Even if you just mix a dsDNA with a histone octamer, under physiological
A. Chromosomal integration occurs during mitosis conditions, the mixture will rearrange to form the Beads-on-Strings structure
B. In chromosomal integration and sister chromatids exchange, crossing- of nucleosomes
over occurs 12. D. Linkers are the “strings” connecting nucleosome beads together
C. Pseudogenes are products of gene conversion. 13. D. On the contrary, it is actually chromatins (“thins”) that make up chromatids
D. Sister chromatids exchange occurs between viral and host DNA 14. E. All statements mentioned are true.
E. Gene rearrangement is involved in the process of IgG synthesis 15. B. Recall, 10nm Fibril and 30nm Chromatin Fibers are the two higher-order
structures for chromatin compaction
30. Which of the following correctly describes Pseudogenes? 16. A. H1 is the stabilizer, and lamins is the anchor site.
A. Products of gene conversion 17. C. Recall that active DNA can have regions hypersensitive to DNase I
B. Processed genes containingnonsense codons 18. C. Enhancers, from the name itself, enhances transcription and is found in the
C. Processed genes containing missense codons active regions of DNA
D. Genes that code for deviated amino acids 19. D. Constitutive chromatin is found near chromosomal centromere and
E. None of the above chromosomal ends
20. C. Recall, Centromeres are marked by A-T rich regions
21. C. Recall, centromeres are bound by H3-variants, CENP-A
ANSWER KEY 22. A. Recall, telomeres are marked by T-G rich region
1. C. Template strand sequence is complementary, not the same, as 23. E. Chromosomes are the most highly-packed structure a DNA can be
mRNA, hence, aka Noncoding organized into
2. C. Complementary DNA strand ang nirerefer, so don’t expect Uracils. 24. D. Alu family members may be mobile elements
Also, if one strand is 3’-5’, automatically, the complement is the reverse 25. C. FraGile X syndrome is due to CGG
or vice versa (5’-3’). Establish that first, saka mo kukunin yung actual 26. D. MyoTonic Dystrophy is due to CTC, not CAG
base complements. 27. B. 16S & 12SmtRNA are encoded by the heavy chain.
3. A. Recall, Histone H1 is the LEAST tightly bound to chromatin. Hence, 28. D. It is gene conversion that homogenizes sequences of members of reps.
easily removed by a salt solution. Very useful in DNA purification 29. E. Recall, genes for light and heavy chains of IgG are distant. Immunoglobulin
techniques gene rearrangement occurs so as to bring them closer to each other.
4. A. Recall, Histone H1 is least tightly bound. Hence, can easily be 30. B. Recall, when a jumping gene (as in transposition) inserts to a segment of
removed. the genome, mutation may occur. However, should the transposition lead to
5. A. H1 lang ang walang oligomer. H2A-H2B forms dimers, while H3-H4 nonsense codons (non-coding; termination codon), the gene is now a Pseudogene
forms tetramers. Recall, Fig 7. H1 is not even part of the core.

Trans By: STH-川端, Landicho, Mortos Page 11 of 11