Mol Bio - Cell cultures, Nucleic Acids, Cell Dogma

complex of cells, which proliferates in vitro and is not organized as tissue.

Cell culture

What time? live cells or tissues isolated from animal

19th century

Who?: established methods of cultivation tissues and cells isolated from adult mammals

Carrel and Burrows

epithelial cells isolated from tissue of various adult animals

1920-1940

1952 —- from malignant human tissue

HeLa cell line

special cultivation techniques -> long term culture

1960 – 1970

• Cell biology during the in vitro condition is___ with the conditions in vivo (in organism)

• Cultivated cells differentiate from others

not comparable

Effect of culture environment on cultivated cells

• characteristics of the substrate the cells grow on (surface of cultivation flask, semisolid gel, solution in suspension cultures etc.); •

extent of contact with other cells

• composition of culture medium (inorganic salts, amino acids, hormones, growth and differentiation factors etc.)

• gas content in the culture system (CO2, O2, etc.)

• incubation temperature.

Conditions for cultivation of cells in vitro

• Sterility

• Temperature

• pH

• Osmotic pressure

• Culture medium

• Serum

• Substrate

Utilization of cell cultures

• Virology

• Pharmacotoxicology

• Human genetics

• Oncology

• Gynecology and Obstetrics

What period of culture – cell adapt to in vitro conditions

Lag phase

What period of culture - cells are proliferating

Logarithmic growth phase (log phase)

What period of culture - cells are living in culture

Stationary phase

What period of culture–cells degenerate and begin to release from the culture

Degradation phase

Type of cell culture? - duration is up to 24 hours or maximum several days

Short term cultures

Type of cell culture? - maintained for more than 10 days

Long term cultures

long chains of nucleotides linked together by phosphodiester bonds

Nucleic acids

building blocks of Nucleic acids

nucleotides

Two types of nucleic acid

• Deoxyribonucleic acid (DNA)

• Ribonucleic acid (RNA)

In eukaryotic cell, DNA is found in the?

nucleus; with small amounts in mitochondria and chloroplasts

In eukaryotic cell, RNA is found in the?

throughout the cell

Nucleic acids are polymers of nucleotides used for:

• storage of genetic info (DNA)

• transmission of genetic info (mRNA)

• processing of genetic information (ribozymes)

• protein synthesis (tRNA and rRNA)

Nucleotides are also used in the monomer form for cellular functions:

• energy for metabolism (ATP)

• enzyme cofactors (NAD+ )

• signal transduction (cAMP)

What nucleic acid? storage of genetic info

DNA

What nucleic acid? transmission of genetic info

mRNA

What nucleic acid? processing of genetic information

ribozymes

What nucleic acid? protein synthesis

tRNA and rRNA

What nucleic acid? energy for metabolism

ATP

What nucleic acid? enzyme cofactors

NAD+

What nucleic acid? signal transduction

cAMP

• “Energy rich” compounds

• Chemical signals

Nucleotides

Nucleotides are composed of 3 components

• Nitrogenous “base”

• Ribose (or deoxyribose)

• Phosphate

Nitrogenous Bases are derivatives of

pyrimidine or purine

Nitrogenous bases contain

heteroaromatic molecules

Nitrogenous base structure

Planar or almost planar structures

Nitrogenous base absorb UV light Absorb around what nm?

250–270 nm

nitrogenous base present in both DNA and RNA

Cytosine, adenine, and guanine

Nitrogenous base present only in DNA

Thymine

Nitrogenous base present only in RNA

Uracil

Purines examples

Adenine(A) and Guanine(G)

Pyrimidine examples

Cytosine(C), Uracil(U), and Thymine (T)

All nitrogenous bases are good of being what?

H-bond donors and acceptors.

Nitrogenous base pH

Neutral molecules at pH 7

MADE OF TWO STRANDS OF POLYNUCLEOTIDE

DNA

The sister strands of the DNA molecule run in opposite directions (____)

• They are joined by the bases

antiparallel

Each base in DNA is paired with a specific partner:
A is always paired with

T

Each base in DNA is paired with a specific partner:

G is always paired with

C

Purine with Pyrimidine

• Thus the sister strands are _____ but not identical

• The bases are joined by ___, individually weak but collectively strong.

complementary; hydrogen bonds

A – T basepair consist of how many h-bonds

2 h-bonds

G – C basepair consist of how many h-bonds

3 h-bonds

One of the most important discoveries in biology

Why is this important? “This structure has novel features which are of considerable biological interest.” ―Watson and Crick, Nature, 1953

Discovery of DNA Structure

Good illustration of science in action

• missteps in the path to a discovery

• value of knowledge

• value of collaboration

• cost of sharing your data too early

Missing layer means alternating pattern (major and minor groove)

– Hydrogen bonding:

A pairs with T

G pairs with C

Double helix fits the data!

Watson and Crick

–“Cross” means helix

–“Diamonds” mean that the phosphatesugar backbone is outside

– Calculated helical parameters

Franklin and Wilkins

Types/forms of DNA: Coding and non-coding DNA

Chromosomal DNA

Types/forms of DNA: Small circular, contains 37 genes and all of which are essential for normal mitochondrial function

Mitochondrial DNA

Types/forms of DNA: structures in the cytosol of the bacteria made of two-stranded circular DNA

Plasmids

• Usually single stranded

• Genetic material of RNA virus

RNA

Functional RNA examples

• e.g. Translation machinery

• rRNA (ribosomal RNA)

• tRNA (transfer RNA)

Regulatory RNA example

Control of gene expression

• miRNA (microRNA)

Gene expression RNA example:

• mRNA (messenger RNA)

• Copy of 1 gene for translation by ribosomes

There are three types of RNA molecules.

• Each has a different function in making or synthesizing proteins.

Messenger RNA (mRNA)

Transfer RNA (tRNA)

Ribosomal RNA (rRNA)

What RNA?
carries DNAs message from the nucleus to the ribosome.

Messenger RNA (mRNA

What RNA?
carries the correct amino acids to the ribosome so they can be added to the growing protein chain

Transfer RNA (tRNA)

What RNA?

makes up part of the ribosome. Helps read mRNAs message and assemble proteins.

Ribosomal RNA (rRNA)

Central dogma:
double strands of DNA splits into two single strands. Each of these single strands acts as a template for a new strand of complementary DNA.

Replication

Central dogma:

DNA is copied (transcribed) to mRNA, which carries the information needed for protein synthesis.

Transcription

Central dogma:
RNA is "reverse transcribed" into DNA and is catalyzed by reverse transcriptase enzymes

Reverse transcription

Central dogma:

process by which mRNA directs protein synthesis with the assistance of tRNA

Translation

There are two steps in protein synthesis, what are they?

1. Transcription

2. Translation

Step of protein synthesis: mRNA is made from the DNA code.

Transcription

Step of protein synthesis:

• It occurs in the nucleus where the DNA is located.

• Involves DNA and mRNA

Transcription

Step of protein synthesis:

Protein is made by adding amino acids to a growing peptide chain.

It occurs at the ribosome where proteins are made.

• Involves mRNA, rRNA and tRNA.

Translation