Manual of Cytogenetics in Reproductive Biology Pankaj Talwar
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1Basics of Genetics and Genetics Laboratory
 
SECTION OUTLINE
  1. Structure of DNA
    • MS Ahuja
  2. Chromosome Nomenclature and cell Division
    • Manisha Vajpayee
  3. Microscopes in Assisted Reproductive Technology
    • Rajvi H Mehta
  4. The Cell Cycle and Cell Division
    • Manisha Vajpayee
  5. Cell
    • Kuldeep Mohanty, Swetasmita Mishra, Kranthi Vemprala, Tarranum Hassan, Rima Dada
2

Structure of DNA1

MS Ahuja
 
INTRODUCTION
Cell is the basic functional and structural unit of life. The functions performed by a cell are influenced both by the information contained in its deoxyribonucleic acid (DNA) and its interaction with the external environment.
Deoxyribonucleic acid is located in two locations in a cell—nucleus and mitochondria. The structure described here refers to nuclear DNA. DNA can be defined as double-stranded macromolecule composed of several nucleotides which are capable of storing genetic information. Each nucleotide, in turn, consists of three types of units: a five-carbon (pentose) sugar (deoxyribose), a nitrogen containing base and a phosphate group (Fig. 1).
The nitrogen bases found in DNA are of two types, purines [i.e. adenine (A) and guanine (G)] and pyrimidines [i.e. thymine (T) and cytosine (C)].
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Fig. 1: Composition of single nucleotide
The nucleotides are joined to each other through phosphodiester bonds between the fifth carbon atom of one deoxyribose (5′) and the third carbon atom of the next deoxyribose (3′). The polynucleotide chain has a sugar-phosphate “backbone” (Fig. 2). The two chains of a DNA molecule wound around each other form a double helical DNA molecule with the nitrogenous bases directed towards the inside of the helix.1
One end of each chain has a terminal sugar residue in which the fifth carbon atom is not linked to any sugar and is called the 5′ end. Similarly, at the other end, the third carbon atom of the terminal sugar molecule is free and is called the 3′ end. One chain runs from the 5′ to the 3′ end, while the other has the opposite orientation and runs from the 3′ to the 5′ end. A nucleotide containing A always pairs with the one containing T, while G always pairs with C (Fig. 3).
These specific purine-pyrimidine couples are called complementary bases and the opposite strands of a DNA molecule are known as the complementary strands. These strands are joined to each other by hydrogen bonds between the bases. There are three hydrogen bonds between C and G, and two between A and T. Though DNA is typically described as being double-stranded, in some viruses DNA is single-stranded also.2
 
DNA PACKAGING
Deoxyribonucleic acid is a long molecule. The total length of DNA [6.6 × 109 base pairs (bp)] in a single human cell is 1 meter. It has to be packed in a nucleus of less than 10 μm size. Thus, it is compactly packed by different levels of coiling. At the first level, the DNA molecule is coiled around proteins called histones to form a bead-like structure known as a nucleosome (Fig. 4).4
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Fig. 2: Two complimentary DNA strands
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Fig. 3: Double helix structure of DNA
At the tertiary level of coiling, chromatin fibers are formed. The chromatin fibers form long loops on a scaffold of nonhistone proteins, which are further wound in a tight coil to make up the chromosome during mitosis. Such compaction of DNA allows its equal distribution to the daughter cells during mitosis. A chromosome consists of equal amounts of DNA, histones and other nonhistone proteins.3
 
FORMS OF DNA
Deoxyribonucleic acid can adopt different types of helical structures. A-DNA and B-DNA are right-handed helices (i.e. the helix spirals in a clockwise direction as it moves away from the observer). They have 11 bp and 10 bp per turn, respectively. Z-DNA is left-handed and has 12 bp per turn. In the majority of cells, B-DNA is present. The width of a DNA molecule (2 nm) is much less than the space left between adjacent turns, creating major and minor grooves.
 
REPLICATION OF DNA
Nuclear DNA replicates before mitosis and also before meiosis I. The DNA content becomes double (4d) as compared to that of a normal diploid cell (2d). The chromosome number of diploid cell is written as 2n. It then becomes compact in the form of visible chromosomes, which are equally distributed to the two daughter nuclei.
Deoxyribonucleic acid replication is semiconservative, both strands of the original DNA molecule are conserved as such and each daughter DNA molecule contains one original and one newly synthesized strand.
Deoxyribonucleic acid replication is initiated at multiple points known as origins of replication. This results in formation of bifurcated, Y-shaped structures known as replication forks (Fig. 5).
This replication then progresses in both directions from these points of origin, forming rounded structures called replication bubbles (Fig. 6).5
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Fig. 4: Stages of DNA packaging
The enzyme DNA helicase separates the two strands. Each strand then synthesizes a complementary strand through the action of DNA polymerases.4
The synthesis of both the strands occurs in the 5′ to 3′ direction. Hence, one strand of the double helix is synthesized in a continuous fashion and is known as the leading strand, while the other is synthesized in pieces called Okazaki fragments. The fragments are joined together by the enzyme DNA ligase to form a continuous strand. This strand is known as the lagging strand.
Deoxyribonucleic acid replication in individual replication units takes place at different times in the synthesis (S) phase of the cell cycle, with adjacent replication units fusing until the entire DNA is copied, forming two identical daughter DNA molecules.4,5
 
RIBONUCLEIC ACID
Genetic information contained in the DNA is transferred from the nucleus to the cytoplasm by ribonucleic acid (RNA) molecules. The chemical structure of RNA is similar to that of DNA, except that the nucleotides in RNA have a ribose sugar instead of deoxyribose, and uracil (U) replaces T as one of the pyrimidines. RNA is usually single-stranded whereas DNA is generally double-stranded.
 
Types of Ribonucleic Acid Molecules
Ribonucleic acid molecules are of three types: messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA).
Ribonucleic acid carrying the coded information from the nucleus to the cytoplasm is known as mRNA. It is synthesized in the nucleus from the DNA template through a process known as transcription. The sequence of an mRNA is the same as that of the gene that transcribes it, except that U replaces T in RNA. The information in the mRNA is used for the synthesis of a polypeptide. This process, known as translation, occurs on ribosomes. Ribosomes are made up of different structural proteins in association with a specialized type of RNA, known as rRNA. Translation involves one more type of RNA, i.e. tRNA which provides the molecular link between the codes base sequence of an mRNA and the amino acid sequence of protein. There is a different tRNA for each amino acid.6
 
GENE STRUCTURE
A gene can be defined as a sequence of bases in a DNA molecule which codes for a polypeptide (Fig. 7). This sequence of bases also includes regulatory sequences. These regulatory sequences are necessary for gene expression, i.e. the production of an mRNA and, in turn, a functional polypeptide. A gene also includes noncoding sequences (introns) that interrupt the coding segments (exons).
Introns alternate with exons and both are transcribed into an mRNA. Although a few genes in the human genome do not have introns, most genes contain at least one and usually several introns. The number of exons is one more than the number of introns. The size of different genes varies from 10 kilobases to thousand of kilobases (kb) (1 kb = 1,000 bases). However, the size of most genes is usually in hundreds ofkb, e.g. the length of the antihemophilic factor VIII gene is 186 kb. Some genes are small, e.g. the length of the beta globin gene is 3 kb, while some are very long, e.g. the dystrophin gene is 2 million bases (mb) (1 mb = 1,000 kb) in length.6
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Fig. 5: Formation of Y-shaped replication forks during DNA replication
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Fig. 6: DNA replication progresses with formation of replication bubbles
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Fig. 7: Diagrammatic representation of gene structure
Theoretically, each of the two complementary DNA strands can code for an mRNA molecule. However, these complementary mRNAs would produce two entirely different proteins. In practice, however, only one of the two strands is required to code for a protein. By convention, this sequence of a gene is referred to as the sense strand or coding strand (strand A in Fig. 7) and the other DNA strand serves as a template for the production of an mRNA and is referred to as the antisense or noncoding strand (strand B in Fig. 7).
 
Untranscribed Portions of a Gene
The nucleotide sequences adjacent to the coding sequence provide the molecular “start” and “stop” signals for the synthesis of an mRNA. At the 5′ end of the gene, immediately upstream from the transcription initiation site, lie untranscribed but important regions. One of these is the promoter region.
This promoter region includes sequences responsible for the proper initiation of transcription. It is usually several hundred nucleotides long and RNA polymerase binds to it. Many promoters often contain a consensus s-equence 5′-TATA-3′ (TATA box), 30–50 bp upstream of the site at which transcription begins.
In addition to promoters, there are other regulatory elements for gene expression such as enhancers, silencers and locus-controlling regions. Some of them may be far away from the gene or even on other chromosomes in the genome. Mutations in the promoter or other regulatory elements can also cause genetic diseases.
At the 3′ end of gene lies an important region that contains the signal for addition of adenosine residues (poly A tail) to the 3’ end of the mRNA. Such closely neighboring regulatory sequences are a part of the gene. However, at present, complete information about regulatory elements spread over the genome is not available and the limits of a gene are still ill-defined.6,7
 
GENE EXPRESSION
 
Transcription
Transcription is the process by which information in a gene is transcribed onto an mRNA. The DNA in the region of the gene to be transcribed uncoils and the two strands separate.
7
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Fig. 8:
The process of transcription. Note that antisense strands of DNA is used as a template for production of mRNA
The antisense strand is used as a template and mRNA is synthesized. The sequence of mRNA is the same as that of the sense strand and complementary to that of the antisense strand (Fig. 8).
Transcription begins with the first exon and proceeds in the 5′ to 3′ direction. Thus, the initiation point corresponds to the 5′ end of the final mRNA product. The initiator (first) codon of any mRNA is always AUG, which codes for methionine. Transcription continues through both the intron and exon portions of the gene. It ends at the position on the chromosome that eventually corresponds to the 3′ end of the mature mRNA.
In eukaryotes, transcription results in the production of pre-mRNA which requires processing to generate a functional mRNA. These post-transcriptional modifications occur in the nucleus and include capping of the 5′ end of the mRNA, addition of a poly A tail at the 3′ end, and removal of introns and splicing of exons to produce the final mRNA. The presence of untranslated regions (UTRs) at both the 5′ and 3′ ends of the molecule is an additional feature of a mature mRNA molecule. The 5′ UTR extends from the capping site to the beginning of protein-coding sequences and can be up to several hundred base pairs in length.
 
Capping
Shortly after the initiation of mRNA transcription, a 7-methylguanosine residue is added to the 5′ end of the primary RNA transcript. This process is called capping. The cap at the 5′ end is characteristic of every mRNA molecule.
 
Splicing of Messenger Ribonucleic Acid
Introns have to be removed before the final mRNA is formed and transported to the cytoplasm (Fig. 9). The exon-intron junctions are marked by specific sequences. At the 5′ exonintron boundary, the intron beings with the sequence GU and ends at the 3′ end with the sequence AG. These sequences, along with the sequences near the splicing site, signal the splicing.
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Fig. 9: Ribonucleic acid splicing resulting in expulsion of intron
Cleavage occurs at both ends of an intron and the exonic mRNA segments are joined together.5,7
 
Translation
The process of synthesis of a polypeptide from an mRNA is known as translation. After the introns are spliced, the mature mRNA is transported from the nucleus to the cytoplasm, where it is translated into a polypeptide chain. In the cytoplasm, tRNA molecules provide a bridge between the mRNA and free amino acids. There are 30 species of tRNA and each one is specific for a single amino acid. Transfer RNAs have a three-nucleotide sequence, which is complementary to the genetic code for a particular amino acid, known as the anticodon. Ribosomes are the site of protein synthesis and mRNA becomes associated with them. Messenger RNA acts as a template for polypeptide synthesis.
 
GENETIC CODE
The sequence of nucleotides in the mRNA decides the order of amino acids in the polypeptide. Three bases code for an amino acid and constitute a codon. The order of codons in a gene is known as the “reading frame”. As there are four types of bases, there can be 64 different combinations of three bases each (triplet). Of these, AUG codes for methionine, which is always the starting amino acid. Three codons (UAA, UAG and UGA) do not code for any amino acid and are known as “stop” or “termination” codons that signal the termination of translation. The remaining codons code for 20 amino acids (Table 1). Abbreviations and symbols of the amino acids are given in Table 2.
Each codon codes for a specific amino acid and there is no overlap. More than one codon can code for the same amino acid and therefore the code is said to be degenerate.88
Table 1   Genetic code
First base
Second base
Third base
U
C
A
G
U
UUU phe
UUC phe
UUA leu
UUG leu
UCU ser
UCC ser
UCA ser
UCG ser
UAU tyr
UAC tyr
UAA stop
UAG stop
UGU cys
UGC cys
UGA stop
UGG tup
U
C
A
G
C
CUU leu
CUC leu
CUA leu
CUG leu
CCU pro
CCC pro
CCA pro
CCG pro
CAU his
CAC his
CAA gln
CAG gln
CGU arg
CGC arg
CGA arg
CGG arg
U
C
A
G
A
AUU ile
AUC ile
AUA ile
AUG met
ACU thr
ACC thr
ACA thr
ACG thr
AAU asn
AAC asn
AAA lys
AAG lys
AGU ser
AGC ser
AGA arg
AGG arg
U
C
A
G
G
GUU val
GUC val
GUA val
GUG val
GCU ala
GCC ala
GCA ala
GCG ala
GAU asp
GAC asp
GAA glu
GAG glu
GGU gly
GGC gly
GGA gly
GGG gly
U
C
A
G
Table 2   Abbreviations for different amino acids
Abbreviation
Name of amino acid
ala (A)
alanine
arg (R)
arginine
asn (N)
asparagine
asp (D)
aspartic acid
cys (C)
cysteine
gln (Q)
glutamine
glu (E)
glutamic acid
gly (G)
glycine
his (H)
histidine
ile (I)
isoleucine
leu (L)
leucine
lys (K)
lysine
met (M)
methionine
phe (F)
phenylalanine
pro (P)
proline
ser (S)
serine
thr (T)
threonine
trp (W)
tryptophan
tyr (Y)
tyrosine
val (V)
valine
 
CONCLUSION
Deoxyribonucleic acid is a macromolecule which stores genetic information. It is typically described as a double helix having two strands running in opposite directions. Each strand or chain is a polymer of subunits called nucleotides. A nucleotide consists of three components—nitrogenous base, five carbon sugar which is called deoxyribose (found in DNA) and ribose (found in RNA) and one or more phosphate groups.
Each strand has a backbone made up of deoxyribose sugar molecules linked together by phosphate groups. The 3′ carbon atom of a sugar molecule is connected through a phosphate group to the 5′ carbon atom of the next sugar. This linkage is also called 3′-5′ phosphodiester linkage. All DNA strands are read from the 5′ to the 3′ end where the 5’ end terminates in a phosphate group and the 3’ end terminates in a sugar molecule.
The DNA molecule is long and to accommodate it into nucleus, it is compactly packed by different levels of coiling. DNA replicates before cell division by semiconservative method.
A gene can be defined as a sequence of bases (nucleotides) in a DNA molecule which codes for a polypeptide. A gene also includes noncoding sequences (introns) that interrupt the coding segments (exons).
The genetic information contained in DNA is transcribed on to mRNA. This mRNA undergoes splicing by which introns 9are removed. It then undergoes translation—the process of synthesis of a polypeptide from an mRNA.
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