MOLECULAR BASIS OF INHERITANCE

- Mendel ke samay, genetic material ki prakriti clear nahin thi.

- DNA - deoxyribonucleic acid - genetic material hai, adhikansh

organisms ke liye.

- Nucleic acids - DNA aur RNA - do prakar ke hote hain.

- DNA genetic material ke roop mein kaarya karta hai, jabki RNA
messenger ke roop mein kaarya karta hai.

- RNA ke anya roles bhi hote hain, jaise ki adapter, structural, aur kuch
cases mein catalytic molecule ke roop mein.

- Is chapter mein DNA ki structure, replication, transcription, genetic

code, protein synthesis (translation), aur unki regulation ki basic baat
karne wale hain.

- Pichhle das saal mein manusya genome ki pooree nucleotide sequence

ka nirdharan kiya gaya hai, jisne genomics ki nayi duniya shuru kar di hai.

- Manusya genome sequencing ke mahatv aur unke parinaamon par baad

mein charcha ki jayegi.

- Sabse pehle, hum DNA ki structure ko samajhenge, jo jeevit pranaliyon

mein sabse adhik rochak anuvanshik hai.

- DNA aur RNA ke beech sambandh par baad mein charcha ki jayegi.

THE DNA
- DNA ek lambi polymer hai jo deoxyribonucleotides se bani hai.
- DNA ki lambayi nucleotides ya base pairs ki sankhya se define hoti
hai.
- Yeh ek organism ki vishishtata hai.
- Udaharan ke liye, φ ×174 bacteriophage mein 5386 nucleotides
hote hain, Bacteriophage lambda mein 48502 base pairs hote hain,
Escherichia coli mein 4.6 × 106 base pairs hote hain, aur manusya
DNA ka haploid sankhya 3.3 × 109 base pairs hota hai.
- Aisi lambi polymer ki structure par hum charcha karenge.

Structure of Polynucleotide Chain

- Ek polynucleotide chain (DNA ya RNA) ki chemical structure mein
ek nucleotide teen hisson se bani hoti hai - ek nitrogenous base, ek
pentose sugar (RNA mein ribose, DNA mein deoxyribose), aur ek
phosphate group.
- Nitrogenous bases do prakar ki hoti hain - Purines (Adenine aur
Guanine) aur Pyrimidines (Cytosine, Uracil aur Thymine).
- Cytosine dono DNA aur RNA mein common hoti hai, Thymine sirf
DNA mein hoti hai, aur Uracil RNA mein Thymine ki jagah hoti hai.
- Ek nitrogenous base, pentose sugar ke 1' C par N-glycosidic
linkage se jude hue, ek nucleoside banati hai.
- Phosphate group aur nucleoside ke 5' C par phosphoester linkage
se jude hue, ek nucleotide (ya deoxynucleotide) banati hai.
- Do nucleotides 3'-5' phosphodiester linkage se jude hue, ek
dinucleotide banate hain.
- Isi tarah adhik nucleotides jude hue, ek polynucleotide chain
banate hain.
- Polymer ke ek end par free phosphate moiety hoti hai, jo 5'-end
kehte hain, aur doosre end par sugar ke 3'C group par free OH hoti
hai, jo 3'-end kehte hain.
- Sugar aur phosphates se backbone banati hai, aur nitrogenous
bases backbone se project karti hain.
- RNA mein har nucleotide residue mein 2' -position par ek
additional -OH group hoti hai.
- RNA mein uracil thymine (5-methyl uracil) ki jagah par payi jati
hai.
- DNA ko pehli baar Friedrich Meischer ne 1869 mein nucleus mein
ek acidic substance ke roop mein identify kiya tha.
- Usne iske liye "Nuclein" naam diya tha.
- Technical limitation ki wajah se DNA ki structure ka pata lagaana
mushkil tha.
- 1953 mein James Watson aur Francis Crick ne Maurice Wilkins aur
Rosalind Franklin dwara produce X-ray diffraction data ke aadhaar
par DNA ki Double Helix model ki proposition di.
- Unki proposition ka ek mahatvapoorn hissa base pairing tha.
- Erwin Chargaff ki observation ke aadhaar par yeh proposition di
gayi thi ki double stranded DNA mein Adenine aur Thymine aur
Guanine aur Cytosine ki ratios constant hoti hain aur ek ke barabar
hoti hain.
- Base pairing se polynucleotide chains ko ek anokhi property milati
hai, jo ki complementary hoti hai.
- Agar ek strand ki base sequence jaani jaati hai, to doosri strand ki
sequence bhi jaani ja sakti hai.
- DNA ki Double-helix structure ki khaasi baatein yeh hain:
- Yeh do polynucleotide chains se bani hoti hai, jahan backbone
sugar-phosphate se bani hoti hai, aur bases andar ki taraf hoti hain.
- Do chains ki polarity anti-parallel hoti hai, yaani ek chain ki
polarity 5'à3' hoti hai, to doosri ki 3'à5' hoti hai.
- Do strands ki bases hydrogen bond (H-bonds) se pair hoti hain,
jisse base pairs (bp) banti hain.
- Adenine aur Thymine doosri strand se do H-bonds se jude hote
hain, aur Guanine aur Cytosine teen H-bonds se jude hote hain.
- Isse purine hamesha pyrimidine ke saamne aata hai, jisse helix
ki do strands ke beech ek saman duri rehti hai.
- Do chains right-handed fashion mein coiled hoti hain, jahan
helix ki pitch 3.4 nm hoti hai, aur har turn mein lagbhag 10 bp hote
hain.
- Base pairs ki plane ek doosre ke upar stack hoti hai, jisse helical
structure ki stability banti hai.
- DNA ki double helix structure ki proposition aur iske genetic
implications ki simplicity ne ek kranti ko janm diya.
- Francis Crick ne jaldi hi molecular biology mein Central dogma ki
proposition ki, jo kehta hai ki genetic information DNA se RNA se
Protein ki taraf bahati hai.
- Yeh Central dogma molecular biology ki mool bhavna hai, jo kehta
hai ki genetic information ki disha ek taraf hoti hai, yaani DNA se
RNA se Protein ki taraf, aur ulta nahi hoti.
- Isne genetic information ki flow ki samajh ko badal diya aur
molecular biology ki research ko ek naye disha mein le gaya.

Packaging of DNA Helix

- DNA double helix ki lambayi ek typical mammalian cell mein 2.2
metres hoti hai, jo ki nucleus ki dimension se kai guna adhik hai.
- Is lambi polymer ko cell mein kaise package kiya jata hai?
- E. coli ki DNA ki lambayi 1.36 mm hoti hai, toh E. coli mein base
pairs ki sankhya kya hogi?
- Prokaryotes jaise E. coli mein, DNA ek defined nucleus mein nahi
hoti, lekin wo cell ke andar scattered nahi hoti.
- DNA (jo negatively charged hoti hai) kuch proteins (jo positively
charged hote hain) ke saath "nucleoid" region mein hoti hai.
- Nucleoid mein DNA badi loops mein proteins ke dwara organised
hoti hai.
- Eukaryotes mein, DNA ki organisation bahut adhik complex hoti
hai.
- Histones ek prakar ke positively charged, basic proteins hote hain.
- Histones mein lysine aur arginine jaise basic amino acid residues
hote hain, jo positive charges lete hain.
- Histones ek unit mein organise hote hain, jise histone octamer
kehte hain.
- Negatively charged DNA, positively charged histone octamer ke
around wrap hoti hai, jisse nucleosome banata hai.
- Ek typical nucleosome mein 200 bp ki DNA helix hoti hai.
- Nucleosomes, chromatin ki repeating unit hote hain, jo nucleus
mein threadlike stained bodies ke roop mein dekhe jaate hain.
- Electron microscope mein, nucleosomes 'beads-on-string'
structure ke roop mein dekhe jaate hain.
- Ek mammalian cell mein kitne nucleosomes (beads) honge, yeh
theori se andaza lagaya ja sakta hai.
- Chromatin ki beads-on-string structure ko chromatin fibers mein
package kiya jata hai, jo metaphase stage mein chromosomes mein
coil aur condense hote hain.
- Chromatin ki packaging ko higher level par karna ke liye
additional proteins ki zaroorat hoti hai, jo Non-histone
Chromosomal (NHC) proteins kehte hain.
- Ek typical nucleus mein, kuch region chromatin loosely packed
hote hain (aur light stain karte hain) aur euchromatin kehte hain.
- Chromatin jo densely packed hota hai aur dark stain karta hai, use
heterochromatin kehte hain.
- Euchromatin transcriptionally active chromatin hota hai, jabki
heterochromatin inactive hota hai.
THE SEARCH FOR GENETIC MATERIAL
- Meischer dwara nuclein ki discovery aur Mendel dwara inheritance
ke principles ki proposition ek hi samay par hui, lekin DNA ko
genetic material ke roop mein siddh karne mein samay laga.
- 1926 tak, genetic inheritance ke mechanism ko khojne ki koshish
molecular level par pahunch gayi thi.
- Gregor Mendel, Walter Sutton, Thomas Hunt Morgan aur kai anya
scientists ki pehli discoveries ne chromosomes ko nucleus mein
sthiti dekhayi, lekin genetic material ka molecule abhi bhi jaana
nahin tha.
- Chromosomes ka molecule kaunsa hota hai, yeh sawal abhi bhi
utha hua tha
Transforming Principle
- 1928 mein, Frederick Griffith ne Streptococcus pneumoniae
(pneumonia ke liye jimmedar bacteria) par ek series of experiments
kiye.
- Usne dekha ki bacteria mein ek ajeeb transformation hua, jismein
ek living organism (bacteria) ne physical form badal di.
- Streptococcus pneumoniae (pneumococcus) bacteria ko culture
plate par grow kiya jaye, to kuch smooth shiny colonies (S) banate
hain, jabki kuch rough colonies (R) banate hain.
- Yeh isliye hota hai kyunki S strain bacteria mein mucous
(polysaccharide) coat hota hai, jabki R strain mein nahi hota.
- Mice ko S strain (virulent) se infect kiya jaye, to ve pneumonia
infection se mar jate hain, lekin R strain se infect kiye jane par ve
pneumonia develop nahi karte.
- Frederick Griffith ne heat-killed S bacteria aur live R bacteria ka
mixture mice mein inject kiya, aur mice mar gaye.
- Usne dead mice se living S bacteria recover kiye.
- Usne nishkarsh nikala ki R strain bacteria ko heat-killed S strain
bacteria dwara kuch "transforming principle" mila hai, jisne R
strain ko smooth polysaccharide coat synthesise karne aur virulent
banne mein madad ki.
- Yeh genetic material ke transfer ke kaaran hua hoga, lekin uske
experiments se genetic material ke biochemical swabhav ka pata
nahi chala.
- Isne genetic material ke concept ko badhava diya, lekin yeh abhi
bhi theori thi.
- Baad mein, Avery, MacLeod aur McCarty ne 1944 mein yeh siddh
kiya ki yeh "transforming principle" DNA hota hai.
Biochemical Characterisation of Transforming Principle
- Oswald Avery, Colin MacLeod aur Maclyn McCarty (1933-44) ke
kaam se pehle, genetic material ko protein mana jata tha.
- Unhone Griffith ke experiment mein "transforming principle" ka
biochemical swabhav khojne ke liye kaam kiya.
- Unhone heat-killed S cells se biochemicals (proteins, DNA, RNA,
etc.) ko pure kiya aur dekha ki kaun sa biochemical live R cells ko S
cells mein transform kar sakta hai.
- Unhone paaya ki sirf DNA hi S bacteria se R bacteria ko transform
kar sakta hai.
- Unhone yeh bhi paaya ki protein-digesting enzymes (proteases)
aur RNA-digesting enzymes (RNases) transformation ko prabhavit
nahi karte, isliye transforming substance protein ya RNA nahi tha.
- DNase ke saath digestion ne transformation ko rok diya, isliye
DNA hi transformation ka kaaran tha.
- Unhone nishkarsh nikala ki DNA hi hereditary material hai, lekin
sabhi biologists isse sahmat nahi the.

The Genetic Material is DNA

- Alfred Hershey aur Martha Chase (1952) ke experiments ne DNA ko
genetic material hone ka sakshat praman diya.
- Unhone bacteriophages ke saath kaam kiya, jo ki bacteria ko infect
karte hain.
- Unhone dekha ki virus ka genetic material bacteria ke andar jata
hai aur bacteria use apne genetic material ke roop mein treat karta
hai.
- Unhone yeh khojne ke liye kaam kiya ki virus se protein ya DNA
bacteria mein jata hai.
- Unhone viruses ko radioactive phosphorus aur radioactive sulfur
ke medium par grow kiya.
- Radioactive phosphorus ke saath grow kiye viruses mein
radioactive DNA tha, lekin radioactive protein nahi tha.
- Radioactive sulfur ke saath grow kiye viruses mein radioactive
protein tha, lekin radioactive DNA nahi tha.
- Radioactive phages ko E. coli bacteria se attach karne diya gaya.
- Phir, infection ke dauran, viral coats ko blender mein agitating
karke bacteria se alag kiya gaya.
- Virus particles ko centrifuge mein spin karke bacteria se alag kiya
gaya.
- Radioactive DNA wale viruses se infect kiye bacteria radioactive
the, isliye DNA hi woh material hai jo virus se bacteria mein jata hai.
- Radioactive protein wale viruses se infect kiye bacteria
non-radioactive the, isliye proteins virus se bacteria mein nahi jate.
- Isliye, DNA hi woh genetic material hai jo virus se bacteria mein
jata hai
Properties of Genetic Material (DNA versus RNA)
- Hershey-Chase experiment ne protein aur DNA ke beech genetic
material ke roop mein debate ko hal kar diya.
- DNA ko genetic material hone ka sakshat praman mil gaya.
- Lekin, kuch viruses mein RNA genetic material hai (jaise Tobacco
Mosaic viruses, QB bacteriophage, etc.).
- DNA aur RNA ke chemical structure mein antar se yeh samajh aaya
ki DNA genetic material kyon hai aur RNA dynamic functions karta
hai.
- DNA aur RNA ke beech do chemical antar hain:
1. DNA mein thymine hota hai, jabki RNA mein uracil hota hai.
2. RNA mein 2'-OH group hota hai, jo ki DNA mein nahi hota.
- Genetic material hone ke liye kuch criteria hain:
1. Replication: Genetic material ko apne replica generate karne ki
ability honi chahiye.
 2. Stability: Genetic material chemically aur structurally stable
hona chahiye.
3. Mutation: Genetic material ko slow changes karne ki ability
honi chahiye.
4. Expression: Genetic material ko 'Mendelian Characters' ke roop
mein express karne ki ability honi chahiye.
- DNA aur RNA dono nucleic acids replication, mutation aur
expression kar sakte hain.
- Lekin, DNA RNA se zyada stable hai aur genetic material ke roop
mein behtar hai.
- RNA ki instability aur catalytic nature ki wajah se yeh labile aur
easily degradable hai.
- DNA mein thymine ki presence additional stability deta hai.
- RNA genetic information ko transmit karne ke liye behtar hai,
jabki DNA genetic information ko store karne ke liye behtar hai.

RNA WORLD
- RNA pehla genetic material tha.
- Abhi bohot sare evidence hain jo suggest karte hain ki essential
life processes (jaise metabolism, translation, splicing, etc.) RNA ke
around evolve hue hain.
- RNA genetic material ke saath-saath catalyst bhi tha (kuch
important biochemical reactions jo RNA catalysts dwara catalyzed
hote hain, protein enzymes dwara nahi).
- Lekin, RNA catalyst hone ke kaaran reactive aur unstable tha.
- Isliye, RNA se DNA evolve hua hai, chemical modifications ke saath
jo ise zyada stable banate hain.
- DNA double-stranded hone ke kaaran aur complementary strand
ke kaaran changes ko resist karta hai, repair process evolve karke.

REPLICATION
- Watson aur Crick ne DNA ke double helical structure ko propose
karte hi, DNA ki replication ke liye ek scheme propose kiya.
- Unke original statement mein kaha gaya hai: "It has not escaped
our notice that the specific pairing we have postulated immediately
suggests a possible copying mechanism for the genetic material"
(Watson aur Crick, 1953).
- Scheme mein kaha gaya hai ki do strands alag ho jayenge aur new
complementary strands ke synthesis ke liye template ka kaam
karenge.
- Replication ke baad, har DNA molecule mein ek parental aur ek
newly synthesised strand hoga.
- Is scheme ko semiconservative DNA replication kaha gaya hai.
- Yeh scheme DNA ki replication ke liye ek mukhya theory hai, jo ki
DNA ke genetic material hone ke liye zaroori hai
The Experimental Proof
- Matthew Meselson aur Franklin Stahl ne 1958 mein ek experiment
kiya jisse DNA ki semiconservative replication ko prove kiya gaya.
- Unhone E. coli ko 15NH4Cl (15N is heavy isotope of nitrogen)
containing medium mein grow kiya, jisse 15N DNA mein incorporate
ho gaya.
- Heavy DNA molecule ko normal DNA se CsCl density gradient mein
centrifugation karke alag kiya gaya.
- Phir unhone cells ko normal 14NH4Cl containing medium mein
transfer kiya aur samples ko various time intervals par extract kiya.
- DNA ko double-stranded helices ke roop mein extract kiya gaya
aur CsCl gradients mein separate kiya gaya.
- Density gradients se DNA ki densities ko measure kiya gaya.
- Results ne dikhaya ki DNA semiconservative replication ke
through replicate hota hai, jismein ek parental strand aur ek newly
synthesised strand hota hai.
- Yeh experiment ne DNA replication ki mechanism ko samajhne
mein madad ki.
- Experiment mein, DNA ko 15N se 14N medium mein transfer karne
ke baad, ek generation ke baad (20 minutes mein), DNA ka density
hybrid ya intermediate tha.
- Doosre generation ke baad (40 minutes mein), DNA ko equal
amounts mein hybrid DNA aur 'light' DNA mila.
- Agar E. coli ko 80 minutes tak grow karne diya jaye, to light aur
hybrid densities DNA molecule ka proportion kya hoga?
- Utar: 80 minutes ke baad, DNA mein 75% light DNA aur 25%
hybrid DNA hoga.
- Taylor aur unke colleagues ne 1958 mein Vicia faba (faba beans)
par radioactive thymidine ka use karke chromosomes mein newly
synthesised DNA ki distribution ko detect kiya.
- Experiments ne prove kiya ki chromosomes mein DNA bhi
semiconservative replication ke through replicate hota hai
The Machinery and the Enzymes
- Main enzyme DNA-dependent DNA polymerase hota hai, jo DNA
template ka use karke deoxynucleotides ki polymerisation ko
catalyse karta hai.
- Ye enzymes highly efficient hote hain, kyunki unhein short time
mein large number of nucleotides ki polymerisation karne ki
zaroorat hoti hai.
- E. coli mein replication process 18 minutes mein complete hota
hai, jismein average rate of polymerisation approximately 2000 bp
per second hota hai.
- Polymerases ko fast aur accurate hona chahiye, kyunki mistakes
during replication mutations ko result kar sakte hain.
- Replication process energetically expensive hota hai, aur
deoxyribonucleoside triphosphates dual purposes serve karte hain -
substrates aur energy provider.
- Additional enzymes ki zaroorat hoti hai replication process ko
complete karne ke liye.
- Replication fork mein replication hota hai, aur DNA-dependent
DNA polymerases polymerisation ko 5'à3' direction mein catalyse
karte hain.
- Discontinuously synthesised fragments ko DNA ligase enzyme join
karta hai.
- DNA polymerases apne aap replication process ko initiate nahi kar
sakte, aur replication randomly initiate nahi hota hai.
- Origin of replication hota hai, jahan se replication originates.
- Vectors provide origin of replication, aur recombinant DNA
procedures mein zaroori hote hain.
- Eukaryotes mein, DNA replication S-phase of cell-cycle mein hota
hai, aur replication aur cell division cycle ko highly coordinated
hona chahiye.
TRANSCRIPTION
- DNA ki ek strand se genetic information ko RNA mein copy karne
ka process transcription kaha jata hai.
- Transcription mein bhi complementarity ka principle follow kiya
jata hai, lekin adenosine ab uracil ke saath base pair forma hai,
thymine ke saath nahi.
- Replication ke ulat, jahan total DNA duplicate hota hai,
transcription mein sirf ek segment aur ek strand copy hota hai.
- Isliye, boundaries define karne ki zaroorat hoti hai jo region aur
strand ko demarcate kare jo transcribe hoga.
- Transcription mein dono strands ko copy nahi kiya jata hai, kyun
ki:
 1. Dono strands alag-alag RNA molecules code karenge, jo
proteins ke liye alag-alag amino acid sequences denge.
2. Do RNA molecules ek dusre ke complementary honge, aur
double-stranded RNA forma honge, jo translation ko rok dega.
- Isliye, transcription mein sirf ek strand ko copy kiya jata hai, jo
genetic information ko accurately transfer karne mein madad karta
hai
Transcription unit
- Ek transcription unit mein teen regions hote hain:
1. Promoter
2. Structural gene
3. Terminator
- Structural gene mein, do strands ko define karne ka ek convention
hota hai:
- Template strand: 3'→5' polarity wala strand, jo RNA polymerase
ke liye template ka kaam karta hai.
- Coding strand: 5'→3' polarity wala strand, jo RNA ke sequence ko
define karta hai (thymine ke sthaan par uracil hota hai).
- Coding strand ko reference point maana jata hai, jabki yeh strand
kuch code nahi karta.
- Yeh convention transcription unit ko define karne mein madad
karta hai.
3' -ATGCATGCATGCATGCATGCATGC-5' Template Strand 5'
-TACGTACGTACGTACGTACGTACG-3' Coding Strand

Transcription Unit and the Gene

- Ek gene ko inheritance ka functional unit ke roop mein define kiya
jata hai.
- Genes DNA par hote hain, lekin unhein DNA sequence ke terms
mein define karna mushkil hai.
- tRNA ya rRNA molecule ke liye DNA sequence bhi ek gene ko define
karta hai.
- Cistron ko ek polypeptide ke liye DNA segment ke roop mein
define kiya jata hai, aur structural gene ko monocistronic
(eukaryotes mein) ya polycistronic (bacteria mein) ke roop mein
samjha ja sakta hai.
- Eukaryotes mein, monocistronic structural genes interrupted
coding sequences hote hain - genes split hote hain.
- Coding sequences ko exons ke roop mein define kiya jata hai, jo
mature RNA mein hote hain.
- Exons ko introns dwara interrupt kiya jata hai, jo mature RNA
mein nahi hote.
- Split-gene arrangement gene ki definition ko aur complicated
bana deta hai.
- Inheritance ek character ko promoter aur regulatory sequences bhi
affect karte hain, isliye regulatory sequences ko loosely regulatory
genes ke roop mein define kiya jata hai, bhale hi ve kisi RNA ya
protein ke liye code nahi karte.
Types of RNA and the process of Transcription
- Bacteria mein teen pramukh prakaar ke RNAs hote hain: mRNA
(messenger RNA), tRNA (transfer RNA), aur rRNA (ribosomal RNA).
- In teenon RNAs ki zaroorat hai ek protein ko synthesize karne ke
liye.
- mRNA template provide karta hai, tRNA amino acids laata hai aur
genetic code ko read karta hai, aur rRNAs translation ke dauran
structural aur catalytic role play karte hain.
- Ek single DNA-dependent RNA polymerase hota hai jo bacteria
mein sabhi prakaar ke RNAs ki transcription ko catalyse karta hai.
- RNA polymerase promoter se bind hota hai aur transcription ko
initiate karta hai (Initiation).
- Ye nucleoside triphosphates ko substrate ke roop mein use karta
hai.

Note: Yeh points bacteria mein RNA ki bhoomika aur RNA

polymerase ki zaroorat ko samajhne mein madad karte hain.
 - RNA
polymerase nucleoside triphosphates ko substrate ke roop mein use
karta hai aur template-dependent fashion mein polymerises karta
hai, complementarity ke rule ko follow karta hai.
- Ye helix ko kholne mein madad karta hai aur elongation ko
continue karta hai.
- Sirf ek short stretch of RNA enzyme se bound rehta hai.
- Jab RNA polymerase terminator region tak pahunchta hai, nascent
RNA aur RNA polymerase dono fall off ho jate hain, jisse
transcription ki termination hoti hai.
- RNA polymerase initiation, elongation aur termination ke teenon
steps ko kaise catalyse karta hai, yeh ek intriguing question hai.
- RNA polymerase sirf elongation process ko catalyse kar sakta hai,
lekin ye initiation-factor (σ) aur termination-factor (ρ) se associate
hokar initiation aur termination ko catalyse kar sakta hai.
- Bacteria mein, mRNA ko active hone ke liye kisi processing ki
zaroorat nahi hoti, aur transcription aur translation ek hi
compartment mein hote hain, isliye translation mRNA fully
transcribed hone se pehle shuru ho sakta hai.
- Eukaryotes mein, do additional complexities hote hain:
1. Nucleus mein kam se kam teen RNA polymerases hote hain
(organelles mein paye jane wale RNA polymerase ke alawa).
 2. RNA polymerase I rRNAs ko transcribes karta hai.

- Eukaryotes mein, teen RNA polymerases hote hain:

1. RNA polymerase I: rRNAs (28S, 18S, aur 5.8S) ko transcribes
karta hai.
2. RNA polymerase III: tRNA, 5srRNA, aur snRNAs (small nuclear
RNAs) ko transcribes karta hai.
3. RNA polymerase II: precursor of mRNA, heterogeneous nuclear
RNA (hnRNA) ko transcribes karta hai.
- Primary transcripts mein exons aur introns hote hain, jo
non-functional hote hain.
- Isliye, splicing process hota hai, jahan introns ko remove kiya jata
hai aur exons ko defined order mein join kiya jata hai.
- hnRNA ko additional processing ki zaroorat hoti hai, jaise ki
capping aur tailing.
- Capping mein, unusual nucleotide (methyl guanosine
triphosphate) ko 5'-end par add kiya jata hai.
- Tailing mein, adenylate residues (200-300) ko 3'-end par add kiya
jata hai, template-independent manner mein.
- Fully processed hnRNA, ab mRNA ke roop mein, nucleus se bahar
transport hota hai aur translation ke liye taiyar hota hai.
- In complexities ka significance ab samajhne lagta hai:
- Split-gene arrangements genome ki ancient feature hai.
- Introns ki presence antiquity ki yaad dilaati hai.
- Splicing process RNA-world ki dominance ko darshaata hai.
- RNA aur RNA-dependent processes ki understanding ab zyada
important hai.
- Translation process mein nucleic acid se amino acid polymer
banane ki zaroorat hoti hai.
- Genetic code ki zaroorat thi jo amino acid sequences ko direct kar
sake.
- George Gamow ne suggest kiya ki 3 nucleotides ka code hona
chahiye, taaki saare 20 amino acids ke liye code ban sake.
- Isse 64 codons generate honge, jo ki zaroorat se zyada hain.
- Gamow ki idea ne further research ko spark kiya, aur genetic code
ki triplet nature aur har amino acid ke liye specific codons ki
identification ko lead kiya.
- Codon ki triplet nature ka proof dene mein zyada mushkil tha.
- Har Gobind Khorana ne chemical method develop kiya, jisse RNA
molecules ko defined combinations mein synthesize kiya ja sakta
tha.
- Marshall Nirenberg ki cell-free system ne protein synthesis ko
samajhne mein bahut madad ki.
- Severo Ochoa enzyme ne RNA ko define sequences mein
polymerise karne mein madad ki.
- Aakhir mein, genetic code ka checker-board taiyar kiya gaya.
 -
Genetic code ki kuch important features hain:
- Codon triplet hota hai.
- 61 codons amino acids ke liye code karte hain, aur 3 codons kisi
amino acid ke liye code nahi karte, isliye ve stop codons ke roop
mein kaam karte hain.
- Kuch amino acids ko ek se zyada codons code karte hain, isliye
code degenerate hai.
- Codon ko mRNA mein contiguous fashion mein padha jata hai,
koi punctuations nahi hote.
- Code nearly universal hai, matlab ki bacteria se lekar human tak
UUU Phenylalanine (phe) ke liye code karta hai.
- AUG ko dual functions hain, ye Methionine (met) ke liye code
karta hai aur initiator codon ke roop mein bhi kaam karta hai.
- UAA, UAG, UGA stop terminator codons hain.
Mutations and Genetic Code
- Gene aur DNA ke relationships ko mutation studies se best samajh
mein aata hai.
- Mutation ke effects ko Chapter 4 mein padha gaya hai.
- Large deletions aur rearrangements ke effects ko samajhna asaan
hai, ye gene ke loss ya gain aur uske function par impact kar sakta
hai.
- Point mutations ke effects ko yaha explain kiya jayega.
- Ek classical example hai beta globin chain ke gene mein single
base pair ki change, jisse amino acid residue glutamate se valine
mein change hota hai, aur ye sickle cell anemia naam ki bimari ka
karan banta hai.
- Structural gene mein base ka insertion ya deletion karne wale
point mutations ke effects ko simple example se samajh mein aata
hai.

- Is exercise se ek conclusion nikalta hai:

- Ek ya do bases ka insertion ya deletion reading frame ko us point
se change kar deta hai.
- Aise mutations ko frameshift insertion ya deletion mutations
kehte hain.
- Teen ya uske multiple bases ka insertion ya deletion ek ya
multiple codon ko insert ya delete kar deta hai, aur reading frame us
point ke baad unaltered rehta hai.
tRNA– the Adapter Molecule
- Francis Crick ne code ki proposition ke shuruaat se hi samajh liya
tha ki code ko read karne aur amino acids se link karne ke liye ek
mechanism hona chahiye.
- Usne adapter molecule ki presence postulate ki, jo code ko read
karega aur specific amino acids se bind karega.
- tRNA, jo pehle sRNA (soluble RNA) kehte the, code ki proposition
se pehle se hi jaana jaata tha.
- Lekin, uski adapter molecule ke roop mein role ko bahut baad
mein assign kiya gaya.
- tRNA mein anticodon loop hota hai jo code ke bases ko
complementary deta hai, aur amino acid acceptor end hota hai jahan
wo amino acids se bind hota hai.
- tRNAs har ek amino acid ke liye specific hote hain
- Initiation ke liye, ek alag specific tRNA hota hai jo initiator tRNA
kehte hain.
- Stop codons ke liye koi tRNAs nahi hote.
- tRNA ki secondary structure ko clover-leaf ki tarah dikhaaya gaya
hai, lekin actual structure mein tRNA ek compact molecule hota hai
jo inverted L ki tarah dekhta hai.

TRANSLATION
- Translation ek process hai jisme amino acids ko polypeptide mein
polymerise kiya jata hai.
- Amino acids ke order aur sequence mRNA mein bases ke sequence
se define hote hain.
- Amino acids ko peptide bond se join kiya jata hai, jo energy require
karta hai.
- Peptide bond formation ke liye, pehle phase mein amino acids ko
ATP ke presence mein activate kiya jata hai aur unhe cognate tRNA
se link kiya jata hai.
- Ribosome cellular factory hai jo proteins ko synthesise karta hai.
- Ribosome mein structural RNAs aur 80 different proteins hote
hain.
- Ribosome ki inactive state mein, wo do subunits mein hota hai -
large subunit aur small subunit.
- Small subunit ko mRNA milne par, translation process shuru hota
hai.
- Large subunit mein do sites hote hain jahan subsequent amino
acids bind hote hain aur peptide bond formation ko favour karte
hain.
- Ribosome peptide bond formation ke liye catalyst ke roop mein
kaam karta hai.
- Translational unit mRNA mein sequence hota hai jo start codon
(AUG) aur stop codon se flanked hota hai aur polypeptide ke liye
code karta hai.
- mRNA mein additional sequences hote hain jo translate nahi hote
aur untranslated regions (UTR) kehte hain.

REGULATION OF GENE EXPRESSION

- Gene expression ki regulation ek broad term hai jo various levels
par ho sakti hai.
- Gene expression ke result mein polypeptide ki formation hoti hai,
isliye isko several levels par regulate kiya ja sakta hai.
- Eukaryotes mein, regulation (i) transcriptional level par (primary
transcript ki formation), (ii) processing level par (splicing ki
regulation), (iii) mRNA ko nucleus se cytoplasm mein transport
karne par, aur (iv) translational level par exert ki ja sakti hai.
- Cell mein genes ko specific function ya functions ke liye express
kiya jata hai.
- Udaharan ke liye, E. coli mein beta-galactosidase enzyme ki
synthesis lactose ko galactose aur glucose mein hydrolyse karne ke
liye hoti hai.
- Bacteria ko lactose ki zarurat nahi hai to ve beta-galactosidase
enzyme ki synthesis band kar dete hain.
- Isliye, metabolic, physiological ya environmental conditions gene
expression ko regulate karte hain.
- Embryo se adult organisms mein development aur differentiation
bhi gene expression ki coordinated regulation se hota hai.
- Prokaryotes mein, transcriptional initiation ki rate ka control
gene expression ko regulate karne ka predominant site hai.
- Transcription unit mein, RNA polymerase ki activity promoter ke
saath interaction se regulate hoti hai.
- Regulatory proteins activators aur repressors ke roop mein kaam
karte hain.
- Prokaryotic DNA ke promoter regions ki accessibility proteins ke
saath interaction se regulate hoti hai.
- Operator region promoter elements ke paas hota hai aur repressor
protein se bind hota hai.
- Har operon ke liye specific operator aur specific repressor hota
hai.
The Lac operon
- Lac operon ki samajh Francois Jacob aur Jacque Monod ke
saath-saath kaam karne se possible hui.
- Ve pehle scientist the jo ek transcriptionally regulated system ki
samajh mein aaye.
- Lac operon mein ek polycistronic structural gene ko common
promoter aur regulatory genes dwara regulate kiya jata hai.
- Aise arrangement bacteria mein bahut common hain aur operon
kehte hain.
- Kuch examples hain lac operon, trp operon, ara operon, his
operon, val operon, etc.
- Lac operon mein ek regulatory gene (i gene) aur teen structural
genes (z, y, aur a) hote hain.
- i gene lac operon ke repressor ko code karta hai.
- z gene beta-galactosidase (β-gal) ko code karta hai, jo lactose ko
galactose aur glucose mein hydrolyse karta hai.
- y gene permease ko code karta hai, jo cell ki permeability ko
β-galactosides ke liye badhata hai.
- a gene transacetylase ko encode karta hai.
- Lac operon ke teenon gene products lactose ki metabolism ke liye
zaroori hote hain.
- Most other operons mein bhi, operon mein present genes same ya
related metabolic pathway mein kaam karne ke liye zaroori hote
hain
- Lactose beta-galactosidase ke substrate hai aur operon ko switch
on/off karne ke liye regulate karta hai, isliye isko inducer kehte
hain.
- Glucose jaise preferred carbon source ki absence mein, agar
lactose growth medium mein diya jaye, toh lactose permease ke
action dwara cells mein transport hota hai.
- Lactose phir operon ko niche di gayi manner mein induce karta
hai.
- Repressor protein i gene se synthesise hota hai aur operator
region se bind karke RNA polymerase ko operon transcribe karne se
rokta hai.
- Inducer ki presence mein, repressor inducer se interact karke
inactivate ho jata hai, jisse RNA polymerase promoter tak pahunchta
hai aur transcription hota hai (Figure 5.14).
- Essentially, lac operon ki regulation bhi enzyme synthesis ko
substrate dwara regulate karne ke tarah samajha ja sakta hai.
- Glucose ya galactose lac operon ke liye inducer nahi ho sakte.
- Aap soch sakte hain ki lactose ki presence mein lac operon kitne
samay tak express hota rahega?
- Lac operon ki regulation repressor dwara negative regulation
kehte hain.

HUMAN GENOME PROJECT

- Pichhle sections mein aapne seekha ki DNA mein base ki sequence
hi genetic information ko determine karti hai.
- Iska matlab hai ki ek organism ya individual ka genetic make-up
uski DNA sequences mein hota hai.
- Agar do individuals alag hote hain, toh unki DNA sequences bhi
alag honi chahiye, kam se kam kuch jagahon par.
- In assumptions ne human genome ki complete DNA sequence ko
find karne ki zarurat paida ki.
- Genetic engineering techniques ki establishment aur DNA
sequences ko determine karne ki simple aur fast techniques ki
availability ne human genome sequencing ka ek ambitious project
shuru kiya gaya 1990 mein.
- Human Genome Project (HGP) ko mega project kaha gaya.
- Project ki magnitude aur requirements ko samajhne ke liye,
project ki aims ko niche define kiya ja sakta hai:
- Human genome mein approximately 3 x 10^9 bp hote hain.
- Agar sequencing ki cost US $ 3 per bp hoti (estimated cost in the
beginning), toh total estimated cost 9 billion US dollars hoga.
- Agar obtained sequences ko typed form mein books mein store
kiya jaye, aur har page par 1000 letters aur har book mein 1000
pages hote hain, toh 3300 books ki zarurat hogi single human cell ki
DNA sequence ko store karne ke liye.
- Data ki enormous amount ne high speed computational devices ki
zarurat paida ki data storage, retrieval, aur analysis ke liye.
- HGP bioinformatics naam ki new area mein rapid development se
closely associated tha.
GOALS OF HGP
- HGP ke kuch important goals yeh the:
- Human DNA mein approximately 20,000-25,000 genes ko
identify karna.
 - 3 billion chemical base pairs ki sequences ko determine karna.
- Is information ko databases mein store karna.
- Data analysis ke liye tools ko improve karna.
- Related technologies ko other sectors, jaise industries, mein
transfer karna.
- Project se related ethical, legal, aur social issues (ELSI) ko
address karna.
- Human Genome Project ek 13 saal ka project tha jo U.S.
Department of Energy aur National Institute of Health dwara
coordinated kiya gaya tha.
- Project ke early years mein, Wellcome Trust (U.K.) ek major
partner ban gaya; additional contributions Japan, France, Germany,
China, aur others se aaye.
- Project 2003 mein complete hua.
- DNA variations ke effects ko samajhne se revolutionary new ways
nikal sakte hain diagnose, treat, aur someday prevent karne ke liye
thousands of disorders ko jo human beings ko affect karte hain.
- Human biology ko samajhne ke liye clues provide karne ke alawa,
non-human organisms ki DNA sequences ko samajhne se unki
natural capabilities ko samajhne mein madad mil sakti hai jo health
care, agriculture, energy production, environmental remediation
mein challenges ko solve karne ke liye apply ki ja sakti hai.
- Kuch non-human model organisms, jaise bacteria, yeast,
Caenorhabditis elegans, Drosophila, plants (rice aur Arabidopsis),
etc., ko bhi sequence kiya gaya hai.
Methodologies - Do major approaches mein methods involve
kiye gaye the.
- Ek approach mein RNA ke roop mein express hone wale genes ko
identify kiya gaya (Expressed Sequence Tags (ESTs) kehte hain).
- Dusre approach mein poore genome ki sequencing ki gayi, jo
coding aur non-coding sequence dono ko contain karta tha, aur phir
sequence mein different regions ko functions assign kiye gaye
(Sequence Annotation kehte hain).
- Sequencing ke liye, cell se total DNA isolate kiya gaya aur relatively
chhote sizes ke random fragments mein convert kiya gaya (recall
DNA ek bahut lamba polymer hai, aur technical limitations hain very
long pieces of DNA ko sequence karne mein).
- Fragments ko suitable host mein clone kiya gaya, jisse unki
amplification ho sakti thi.
- Cloning ke liye commonly used hosts bacteria aur yeast the, aur
vectors ko BAC (bacterial artificial chromosomes) aur YAC (yeast
artificial chromosomes) kehte hain.
- Fragments ko automated DNA sequencers se sequence kiya gaya,
jo Frederick Sanger dwara develop kiye gaye method par work karte
the.
- Sequences ko overlapping regions ke aadhar par arrange kiya
gaya.
- Alignment ke liye specialised computer-based programs develop
kiye gaye.
- Sequences ko subsequently annotate kiya gaya aur unhe each
chromosome mein assign kiya gaya.
- Chromosome 1 ki sequence May 2006 mein complete ki gayi (ye 24
human chromosomes - 22 autosomes aur X aur Y - mein se last thi
jo sequence ki gayi).
- Ek aur challenging task genetic aur physical maps ko genome par
assign karna tha.
- Ye polymorphism ke information ka use karke generate kiya gaya,
jisme restriction endonuclease recognition sites aur repetitive DNA
sequences kehte hain microsatellites (repetitive DNA mein
polymorphism ka ek application).
Salient Features of Human Genome
Some of the salient observations drawn from human genome
project are as follows:
(i) The human genome contains 3164.7 million bp.
(ii) The average gene consists of 3000 bases, but sizes vary greatly,
with the largest known human gene being dystrophin at 2.4 million
bases.
(iii) The total number of genes is estimated at 30,000–much lower
than previous estimates of 80,000 to 1,40,000 genes. Almost all
(99.9 per cent) nucleotide bases are exactly the same in all people.
(iv) The functions are unknown for over 50 per cent of the
discovered genes.
(v) Less than 2 per cent of the genome codes for proteins.
 (vi) Repeated sequences make up very large portion of the human
genome.
(vii) Repetitive sequences are stretches of DNA sequences that are
repeated many times, sometimes hundred to thousand times. They
are thought to have no direct coding functions, but they shed light
on chromosome structure, dynamics and evolution.
(viii) Chromosome 1 has most genes (2968), and the Y has the
fewest (231).
(ix) Scientists have identified about 1.4 million locations where
singlebase DNA differences (SNPs – single nucleotide
polymorphism, pronounced as ‘snips’) occur in humans. This
information promises to revolutionise the processes of finding
chromosomal locations for disease-associated sequences and
tracing human history.
Applications and Future Challenges
- DNA sequences se meaningful knowledge derive karna aane wale
decades mein research ko define karega, jo humein biological
systems ki samajh mein madad karega.
- Ye enormous task tens of thousands of scientists ki expertise aur
creativity ko require karega, jo varied disciplines mein public aur
private sectors mein worldwide kaam karte hain.
- HG sequence ke sabse bade impact mein se ek radically new
approach to biological research ho sakta hai.
- Pehle, researchers ne ek ya few genes ko study kiya.
Whole-genome sequences aur new high-throughput technologies
ke saath, hum questions ko systematically aur broader scale par
approach kar sakte hain.
- Ve ek genome ke sabhi genes ko study kar sakte hain, ya ek
particular tissue, organ, ya tumor mein sabhi transcripts ko, ya
kaise tens of thousands of genes aur proteins interconnected
networks mein work karte hain life ki chemistry ko orchestrate
karne ke liye.
- Ye humein biological systems ki deep understanding mein madad
karega aur naye treatments, therapies, aur medicines ko develop
karne mein madad karega.
DNA FINGERPRINTING
- 99.9% base sequence humans mein same hoti hai, isliye 3 × 10^9
bp mein kuch base sequences mein differences hongi.
- Ye differences in DNA sequence hi every individual ko unique
banati hain unki phenotypic appearance mein.
- Agar hum genetic differences ko find karne ke liye DNA
sequencing karte hain, toh ye ek daunting aur expensive task ho
sakta hai.
- DNA fingerprinting ek quick way hai compare karne ke liye DNA
sequences of any two individuals.
- DNA fingerprinting mein repetitive DNA regions mein differences
ko identify kiya jata hai, jahan ek small stretch of DNA ko many
times repeat kiya jata hai.
- Ye repetitive DNA bulk genomic DNA se alag kiye jate hain density
gradient centrifugation ke time.
- Satellite DNA ko micro-satellites, mini-satellites etc. mein
classify kiya jata hai, jo human genome ka large portion form karti
hain.
- Ye sequences normally proteins ko code nahi karti hain, lekin
polymorphism ko show karti hain jo DNA fingerprinting ke basis
hain.
- DNA from every tissue ek individual ke show same degree of
polymorphism, isliye forensic applications mein identification tool
ke roop mein useful hoti hain.
- Polymorphism inheritable hota hai parents se children mein,
isliye DNA fingerprinting paternity testing ke basis hoti hai disputes
ke case mein Polymorphism (variation at genetic level) mutations
ke kaaran hota hai.
- Naye mutations ek individual mein somatic cells ya germ cells
mein aa sakte hain.
- Germ cell mutation agar individual ki reproductive ability ko
seriously impair nahi karta, toh ye mutation population mein spread
ho sakta hai.
- Polymorphism ko genetic mapping aur DNA fingerprinting ke liye
samajhna zaroori hai.
- Allelic sequence variation ko DNA polymorphism kehte hain agar
ek variant (allele) ek locus par human population mein frequency
greater than 0.01 ke saath hota hai.
- Simple terms mein, agar ek inheritable mutation population mein
high frequency mein observed hota hai, toh use DNA polymorphism
kehte hain.
- Noncoding DNA sequence mein variation ki probability zyada hoti
hai kyonki mutations inme immediate effect/impact nahi karta.
- Ye mutations generation ke baad generation accumulate hote hain
aur variability/polymorphism ka basis form karte hain.
- Polymorphisms ki variety hoti hai, single nucleotide change se
lekar very large scale changes tak.
- Evolution aur speciation ke liye, ye polymorphisms bahut
important role play karte hain.
- DNA Fingerprinting ki technique Alec Jeffreys ne develop ki thi.
- Usne satellite DNA ko probe ke roop mein use kiya jo very high
degree of polymorphism show karta hai.
- Isko Variable Number of Tandem Repeats (VNTR) kehte hain.
- Technique mein Southern blot hybridisation ka use kiya gaya tha
radiolabelled VNTR ko probe ke roop mein.
(i) isolation of DNA,
(ii) digestion of DNA by restriction endonucleases
(iii) separation of DNA fragments by electrophoresis,
(iv) transferring (blotting) of separated DNA fragments to synthetic
membranes, such as nitrocellulose or nylon,
(v) hybridisation using labelled VNTR probe, and
(vi) detection of hybridised DNA fragments by autoradiography. A
schematic representation of DNA fingerprinting
- VNTR mini-satellite ke ek class mein aata hai.
- Ek small DNA sequence ko tandemly many copy numbers mein
arrange kiya jata hai.
- Copy number ek individual mein chromosome se chromosome
mein vary karta hai.
- Repeat ki numbers mein polymorphism ka degree bahut zyada
hota hai.
- Isliye, VNTR ki size 0.1 se 20 kb tak vary karti hai.
- Autoradiogram mein many bands of differing sizes aati hain.

- Ye bands ek individual DNA ke liye characteristic pattern deti hain.

- Ye pattern individual se individual mein alag hota hai population
mein, monozygotic (identical) twins ke case mein except.
- Technique ki sensitivity ko polymerase chain reaction (PCR) ka
use karke badhaya gaya hai.
- Isliye, single cell se DNA enough hota hai DNA fingerprinting
analysis ke liye.
- Forensic science mein application ke alawa, iska application
population aur genetic diversities ko determine karne mein bhi hai.
- Abhi, many different probes ka use DNA fingerprints generate
karne ke liye kiya jata hai.