DNA REPLICATION AND REPAIR

The process of DNA duplication is usually called Replication.

The replication is termed “Semiconservative” since each new cell contains one strand of original DNA and one newly synthesized strand of DNA. The original polynucleotide strand of DNA serves as a template to guide the synthesis of the new complementary polynucleotide of DNA.

In a cell, DNA replication begins at specific locations in the genome, called "origins of replication.”

STEPS OF DNA REPLICATION

TELOMERES

• Repetitive sequences at the ends of the linear DNA molecule in eukaryotic chromosomes.
• With each round of replication, these are shortened because DNA polymerase can’t complete synthesis of 5’ end of the each strand.
• This contributes to aging of the cells.

TELOMERASE

      

      

• An enzyme in eukaryotes used to maintain the telomeres.

      

• Has telomerase reverse transcriptase activity.

      

• Present only in embryonic cells, germ cells (reproductive) and stem cells.

SIGNIFICANCE OF TELOMERASE

Cancer cells have high levels of telomerase which prevents telomeres from being short and contribute to immortality of malignant cells.

REVERSE TRANSCRIPTASE

RNA dependant DNA polymerase

Requires RNA template to direct the synthesis of new DNA.

Retroviruses (HIV) have this activity.

COMPARISION OF DNA & RNA SYNTHESIS

DNA REPAIR

Can be damaged by chemicals or radiation

Incorrect bases can also be incorporated during replication.

Multiple repair systems have evolved, allowing cells to maintain the sequence stability of their genomes.

     

1. Excision Endonuclease

     

2. Uracil glycosylase

     

3. AP endonuclease

REPAIR OF THYMINE DIMER

REPAIR OF DEAMINATED & MISSING BASES

Cytosine can become deaminated spontaneously by reaction with nitrous acid to form Uracil

Difference between Inguinal Hernias

Herniation of abdominal viscera can occur in one of several weak aspects of the abdominal wall (e.g. inguinal,femoral , umbilical, or diaphragmatic). Inguinal hernias are the most common of the abdominal hernias and occur more frequently in males due to the inherent weakness of the male inguinal canal. Inguinal hernias occur superior to the inguinal ligament. 

Two types of Inguinal hernias are described :

Indirect inguinal hernias: Indirect hernias result when abdominal contents protrude through the deep inguinal ring lateral to the inferior epigastric vessels. After passing through the inguinal canal and superficial ring, the viscera can  continue and coil in the scrotum. Indirect hernias follow the route taken by the testis and are found with in the spermatic cord. They are covered by the three layers of spermatic fascia.

Direct inguinal hernias: During a direct inguinal hernia, the abdominal contents will protrude through the weak area of the posterior wall of the inguinal canal medial to the inferior epigastric vessels (in the inguinal [Hesselbach's triangle]). Direct hernias rupture through the posterior wall of the inguinal canal and area usually found on the surface of the spermatic cord and bulge at the superficial ring. They may be covered by only the external layer of spermatic fascia.

Note: 

• That both Direct and Indirect hernias may exit through the superficial ring but only Indirect hernias pass through the deep ring.

• Direct hernias are found medial to the inferior epigastric vessels, and Indirect hernias occur lateral to the inferior epigastric vessels.

Clinical correlate

• Inguinal (Hasselbach's) Triangle : 

Direct inguinal hernias usually pass through it

Lateral border: Inferior epigastric vessels

Medial border: Rectus abdominis muscle

Inferior border: Inguinal ligament

• A persistent process vaginalis often results in a congenital indirect inguinal hernia.

• A collection of serous fluid in the tunica vaginalis form a hydrocele resulting in an enlarged scrotum. A hydrocele does not reduce in size when the patient is lying down.

• Inguinal hernias pass above the inguinal ligament 

• Femoral hernias pass below the inguinal ligament.

Femoral hernias: most often occur in women,

TRANSCRIPTION & RNA PROCESSING

TRANSCRIPTION

• Formation of the base sequence of a single stranded molecule of RNA from the base sequence of a dsDNA molecule.
• Only one strand of DNA molecule (i.e, template strand) is copied by RNA polymerase as it synthesize RNA in 5’ to 3’ direction for any particular gene.
• RNA product is antiparallel & complementary to the template strand.
• RNA polymerase recognizes start signals (promoters) and stop signals (terminators).

TYPES OF RNA

Ribosomal RNA (rRNA): most abundant used as structural component of the ribosome associates with ribosomal     proteins to form the complete, functional ribosome.

Transfer RNA (tRNA): second most abundant RNA carry amino acids to the ribosome during protein synthesis.

                                        

Messenger RNA (mRNA): carries information specifying  the amino acid sequence of a protein to the ribosome.Only type of RNA that is translated. 

                                               

Heterogeneous nuclear RNA (hnRNA): found only in the nucleus of the eukaryotic cells.Precursors of mRNA, formed during posttranscriptional processing.

                                                                   

Small nuclear RNA (snRNA): found in eukaryotic nucleus major functional is to participate in splicing mRNA (removal of introns)

Ribozymes: RNA molecules with enzymatic activity. 

Found both in prokaryotes & eukaryotes.

CONCEPTS AND TERMINOLOGY : TRANSCRIPTION

RNA is synthesized by a DNA dependant RNA polymerase.

RNA polymerase locates gene in DNA and binds the promoter site. No primer is required.

RNA polymerase moves along the template strand in the 3’ to 5’ direction as it synthesizes the RNA product in the 5’ to 3’ direction. The RNA product is complementary and antiparallel to the template strand. Coding strand is identical in sequence to RNA product except that RNA contains U instead of T in DNA.

The numbering system is used to identify the location of important bases. The 1st base transcribed as RNA is defined as the +1 base of that gene region. To the left (upstream) of it, bases are -1,-2,-3,etc. To the right (downstream) are +1,+2,+3,etc.

Transcription ends when RNA polymerase reaches a termination signal.

RNA Polymerases 

Production of Prokaryotic mRNA

• The mRNA produced by the gene is a monocistronic in the given figure. That is, it is transcribed from a single gene and codes for only a single protein.
• Some bacterial operons produce polycistronic messages. The mRNA in this case contains information from several genes and codes for several different proteins.

Production of Eukaryotic RNA

Processing of Eukaryotic mRNA

• Alternative Splicing of Eukaryotic mRNA
• For some genes, the primary transcript is spliced differently to produce two or more types of a protein from same gene k/a alternative splicing. E.g : Troponin (T, C, I) Immunoglobulins.

Ribosomal RNA

Transfer RNA (tRNA)

The important regions in tRNA are:

 1. Acceptor arm: appropriate AA is attached.

 2. Anticodon arm: recognizes a specific codon ( for the AA it carries) in mRNA.

The CCA tail is a cytosine-cytosine-adenine sequence at the 3' end of the tRNA molecule. This sequence is important for the recognition of tRNA by enzymes and critical in translation.

Structure of tRNA

Summary:

Recombinant DNA

Overview of rDNA

1.Isolate DNA 

2.Cut with restriction enzymes 

3.Ligate into cloning vector 

4.transform recombinant DNA molecule into host cell 

5.each transformed cell will divide many, many times to form a   colony of millions of cells, each of which carries the recombinant DNA molecule (DNA clone)

Cutting DNA

Restriction enzymes are the scissors of molecular genetics. Restriction enzymes (RE) are endonucleases that will recognize specific nucleotide sequences in the DNA and break the DNA chain at those points. A variety of RE have been isolated and are commercially available. Most cut at specific palindromic sites in

the DNA (sequence that is the same on both antiparallel DNA strands). These cuts can be a staggered which generate “sticky or overhanging ends” or a blunt which generate flush ends.

Joining DNA

Once you have isolated and cut the donor and vector DNAs, they must be joined together. The DNAs are mixed together in a tube. If both have been cut with the same RE, the ends will match up because they are sticky. DNA ligase is the glue of molecular genetics that holds the ends of the DNAs together. DNA ligase creates a phosophodiester bond between two DNA ends.

Amplifying the recombinant DNA

To recover large amounts of the recombinant DNA molecule, it must be amplified. This is accomplished by transforming the recombinant DNA into a bacterial host strain. (The cells are treated with CaCl2 ---> DNA is added ---> Cells are heat shocked at 42 C ---> DNA goes into cell by a somewhat unknown mechanism.)

    Once in a cell, the recombinant DNA will be replicated. When the cell divides, the replicated recombinant molecules go to both daughter cells which themselves will divide later. Thus, the DNA is amplified.

Vectors

Requirements for a cloning vector

a) Should be capable of replicating in host cell

b) Should have convenient RE sites for inserting DNA of interest

c) Should be small and easy to isolate

TYPES:

Expression vectors 

vectors that carry host signals that facilitate the transcription and translation of an inserted gene. They are very useful for expressing eukaryotic genes in bacteria.

Others: Cosmids

              YAC (Yeast Artificial Chromosomes)

              Retroviruses

Why is rDNA important? 

Recombinant DNA has been gaining in importance over the last few years, and recombinant DNA will only become more important in the 21st century as genetic diseases become more prevelant and agricultural area is reduced.  Below  are some of the areas where Recombinant DNA will have an impact.

Better Crops (drought & heat resistance)

Recombinant Vaccines (ie. Hepatitis B)

Prevention and cure of sickle cell anemia

Prevention and cure of cystic fibrosis

Production of clotting factors

Production of insulin

Plants that produce their own insecticides

Germ line and somatic gene therapy

Genetic Testing

Blotting Techniques:

Gel electrophoresis – DNA fragments of different sizes can be separated by an

electrical field applied to a “gel”. The negatively charged DNA migrates away from the

negative electrode and to the positive electrode. The smaller the fragment the faster it

migrates.

POLYMERASE CHAIN REACTION

Short sequence of DNA can be amplified into millions fold within few hours.

Uses of PCR

Diagnosis of various diseases
Genetic fingerprinting
Paternity testing
To detect HIV in newborn whose mother are HIV +ve.
To detect the viral load.

Gene Regulation

Regulation of prokaryotic gene expression

Transcriptional control of prokaryotic can be done by:

a. Regulation by activator and repressor proteins in the lactose operon

b.  Attenuation control in the histidine operon

The Lactose Operon( Lac operon)

The Histidine Operon

Regulation of Eukaryotic Gene Expression

Stimulation of transcription by enhancer and its ass. TF

Transcription Factors

General Transcription Factors

Common to most genes.

TFIID must bind to TATA box before RNA polymerase can bind.

Other e.g: SP-1

             NF-1

Specific Transcription Factors

Binds to enhancer regions, or in a few cases to silencers and modulate the formation of initiation complex, thus regulating rate of initiation of transcription.

E.g: Steroid receptors

       CREB proteins