Genetics Notes

The Central Paradigm - from DNA to RNA to Protein

Genetic information is written as codons. Codons are nucleotides (nitrogenous bases) that are arranged in groups of three (triplet code). Master information is on a DNA molecule. DNA cannot leave the nucleus in eukaryotic organisms. Threre is 20 amino acids and 4 base pairs.

Transcription is the process of making Messenger RNA (mRNA) off DNA. Transcription occurs when information (base sequence) of DNA is transcribed (rewritten) to make single-strand of RNA (messenger RNA). This process must occur in the nucleus because the DNA molecule is to large to pass through the nuclear membrane. The mRNA is able to leave the nucleus so that it can create the necessary proteins in the cytosol. The mRNA is complimentary to the section of DNA it copied. DNA was the template. Uracil replaces Thymine in RNA. RNA nucleotides are linked by transcription enzyme called RNA Polymerase. RNA polymerase must be told where to start and where to stop the transcribing process. The start signal is the promoter area of DNA, the promoter is a sequence of bases called a TATA box. TAC is the 'start' codon which sets the triplet code.

The first phase of transcription is triggered when a speciffic hormone or signal protein enters the nucleus as a message that a speciffic protein section of the DNA is needed. The enzyme RNA polymerase attaches to the promoter of DNA. For any gene, the promoter region signals only one of the two strands to be transcribed. This is the lead strand of DNA, this is where mRNA are made. The other strand of DNA is the lag strand, where tRNA is made. RNA elongates as RNA polymerase assembles it in sequence complimentary to DNA.

Finally RNA polymerase reaches a section on the DNA lead strand called the terminator. The terminator signals the end of the gene and causes the RNA polymerase to detach. The DNA strand reforms a double helix and the mRNA is released.

Genetic messages are translated in the Cytoplasm. Messenger RNA conveys the genetic information from DNA in the nucleus to the translation devices in the cell's cytoplasm. The mRNA and tRNA leaves the nucleus (in eukaryotes) and enters the cytoplasm. The tRNA is a molecular interpretor that translates base sequences to a proper amino acid. In living cells, amino acids are either synthesized or obtained from food. The tRNA picks up the appropriate amino acid and then recognises the appropriate mRNA codon to place the amino acids in the right sequence.

The stop signals on DNA are ACT, ATT, ATC.

The tRNA is a single strand of RNA made of about eighty nucleotides. The tRNA forms several double stranded regions as it bends, folds, and twists, forming hydrogen bonds to maintain its new shape. A single stranded loop at one end of this folded molecule contain a special triplet of bases called the anticodon. The anticodon is complementary to a codon triplet on mRNA. At the other end of the tRNA molecule is a site where an amino acid can attach. A tRNA molecule cannot recognise an amino acid by itself. An enzyme ensures that the appropriate amino acid attaches to a tRNA. There is at least one enzyme for each amino acid. Each enzyme specifically binds one type of amino acid to the appropriate tRNA molecule. A molecule of ATP is required to drive this reaction (binding the amino acid to the tRNA).

There are three stages in the process of translation.

The first stage in the process of translation is initiation. This process is responcible for bringing together the mRNA, tRNA carrying the first amino acid (methionine), and the two ribosomal subunits. First mRNA binds to the small ribosomal subunit. Next an initiator tRNA brings methionine and locaes and binds to the specific codon (start codon [AUG on mRNA]). UAC is anticodon on tRNA (reads TAC on DNA). Then the large ribosomal subunit binds to the smaller one, creating a functional ribosome. The initiator tRNA fits into the 'P' site on the Ribosome.

The second stage in the process of translation is elongation. This process is the addition of amino acids to the polypeptide. First an incoming tRNA/amino acid complex pairs with the next codon on mRNA at the 'A' site of the ribosome. This occurs to enable codon recognition. Next is peptide bond formation. The polypeptide on 'P' site seperates trom tRNA of 'P' site and attaches by peptide bonding to the amino acid carried by tRNA located on the 'A' site. An Enzyme in the ribosome catalyzes the peptide bond formation. Then, translocation occurs. The tRNA in the 'P' site leaves the ribosome (to search for a new amino acid) and the 'A' site bound tRNA, holding polypetide chain is translocated to 'P' site. mRNA and tRNA move as a unit. Elongation continues until a 'stop' codon is reached.

The third stage in the process of translation is Termination. This is when the stop codon (UAA, UAG, UGA) reaches the ribosome's 'A' site. The 'stop' codons do not code for amino acids but signal the end of translation. A complete polypeptide is now freed from the ribosome. The riboome then splits into two subunits (large and small).

Multicellular Eukaryotic organisms undergo cellular differentiation. Cells develop different structures depending upon their function. It is the regulation of genes that leads to this specialization. The nuclei of differentiated cells contains the same genetic potential of undifferentiated cells contain the same genetic potential of undifferentiated cells.

Regeneration is the replacement of lost body parts. This is common in some plants, flatworms, roundworms and salamanders.

Each type of differentiated cell has a particular pattern of expressed genes. Genes are selectively expressed. As an embryo develops and successive cells divide, specific genes are activated at specific times. Cells that preform the same functions are tissues. Different tissues combine to preform the same function in organs. Different organs preform different functions for the same purpose in organ systems. Organ systems preform in synchrony to give life to the organism.

Control of Eukaryotic gene expression at transcription regulates proteins called transcription factors which work together to help RNA polymerase attach to the promoter. DNA sequences called enhancers exist some distance from the genes that they regulate. Activator proteins, one type of transcription factor, bind to the enhancers (DNA sequences). Other transcription factors interact with the activator proteins on the enhancers, bending the DNA, so that now all transcription factors can bind as a complex at the genes promoter. This attachment of transcription factors to both the enhancers and promoter facilitates the correct attachment of RNA polymerase to the promoter to initiate transcription. Repressor proteins may bind to DNA sequences called silencers that then behave like the transportation factors discussed above to inhibit the start of transcription.

The processing of Eukaryotic RNA prior to Translation begins with a cap (guanine nucleotide) and a tail (chain of adenine nucleotides). They Protect mRNA from attack by cellular enzymes and help ribosomes recognize the strand as mRNA. The genes of plants and animals contain non-coding regions within them called introns which are removed from the mRNA before it leaves the nucleus. The coding sections of the genes, called exons are joined by RNA splicing to produce an mRNA molecule with a continuous coding sequence. RNA splicing occurs in two ways. The first way that RNA splices is with mRNA. The mRNA splicing is achieved with a complex of proteins and small RNA molecules. The second way that RNA splicing is achieved is when the RNA transcript itself catalyzes its own splicing as an enzyme. This RNA processing helps control the flow of mRNA from the nucleus to cytoplasm. Sometimes splicing can be carried out in more than one way. This alternative splicing allows translation of more than one polypeptide from a single gene.

There are several regulatory mechanisms for gene expression in Eukaryotes.

DNA packing is a regulatory mechanism for gene expression in Eukaryotes. In DNA packing, each chromosome is a single DNA molecule, and is a very long DNA strand. The DNA double helix is attached to histones (small proteins) and winds around a group of eight of these histones to form a nucleosome. The beaded DNA string then is coiled into a tight helical fiber. This helical fiber then coils further to form a thick supercoil. The supercoil then loops and folds to form the chromosome.

DNA packing prevents gene expression by making it difficult/impossible for RNA polymerase to contact the DNA. For a gene to be transcribed, the histones must loosen their bonds to the DNA. This action is mediated by other proteins.

Cells may use even higher levels of packing for long-term inactivation of genes. Female mammals have one X chromosome compacted into barr body.

Translation can also be regulated. A process called mRNA breakdown occurs in prokaryotic mRNA which has a short lifetime compared to eukaryotic mRNA which can last weeks. Regulatory proteins in transcription called inhibitors are blocked. The processing of proteins after translation id done by Golgi. The selective breakdown of proteins occurs in response to changes in environment, or removal of damaged and or malfunctioning proteins.

A homeotic gene is a master gene that regulates batteries of other genes that actually create the anatomical identity of the rest of the body. It is the basic body plan and is important evolutionary evidence. Cascades of gene expression are protein products of one set of genes that activated another set of genes.

Cell-to-cell signaling also plays a part in development.

Often, a single molecule attaches to the surface of the cell at a receptor site. This starts a series of molecular changes that converts this attachment signal into a specific response inside the cell. This transformation is called a signal transduction pathway. This signaling, in development, can result in transcription factors being activated, mRNA produced, and the translation and production of necessary protein.

Key developmental genes, homeotic genes, are present in a wide variety of organisms. All homeotic organisms have very similar sequences – shows its importance and ancient origin. All homeotic genes have a common sequence of 180 nucleotides called homeoboxes. The polypeptide made by the homeobox binds to specific sequences in DNA, turning “on” and “off” genes during development.

Cancer can result from mutation or changes in the way some genes are expressed.

A gene that causes cancer is an oncogene. Some viruses carry oncogenes that they insert into host cell DNA, making the host cell cancerous.

Human cells contain genes that can be converted to oncogenes. A normal gene with the potential to become an oncogene is called a proto-oncogene.

Many proto-oncogenes code for proteins that stimulate cell division (growth factors). For a proto-oncogene to become an oncogene, a DNA mutation must occur. Most of these mutations occur in somatic cells.

Three changes in somatic cell DNA can produce active oncogenes.

  1. A mutation in a proto-oncogene creates an oncogene that codes for a protein that is hyperactive, stimulating sell growth more actively than normal. The same total amount of protein is produced, however.
  2. An error in DNA replication or recombination generates multiple copies of the gene, all of which are transcribed, producing much more of the normal protein.
  3. Te proto-oncogene is moved to a new site. Under the control of a different promoter that causes it to be transcribed more than normal. The normal protein is then secreted in excess.

In all three cases the cell is stimulated to divide more than normal. Changes in genes that inhibit cell division are also involved in cancer. These genes are tumor suppressor genes, because they normally prevent uncontrolled cell growth.

One type of signal transduction pathway stimulates cell division – a protein is produced by the gene that acts at 62 checkpoint, telling the cell not to divide.

A second type of signal transduction pathway causes the target cell to make proteins that inhibit cell division at 62 checkpoint.

Both types of these interact with components of the cell cycle control system.

Oncogene “hyperactive protein” that is part of the signal transduction pathway may continue to stimulate cell growth after growth factor is no longer present.

A mutant tumor suppressor protein, like a non-functional transcription factor, cannot be activated by the signal transduction pathway. Then the gene that produces the protein that inhibits cell division cannot be made, so the cell keeps dividing. More than somatic mutation is needed to produce a full-fledged cancer cell, but because DNA mutations pass from one generation to the next, the mutations become cumulative until mutations in both oncogenes and tumor-suppressor genes lead to cancer.

DNA technology and the Human Genome:

Even bacteria, which reproduce by binary fission, exchange DNA (through sex pilli). Pieces or copies of pieces of genetic information are exchanged through a cytoplasmic bridge.

Recombinant DNA technology:
Genes from different sources are spliced together in a testube, then introduced into a cell where it can be replicated and even transcribed and translated into a protein.

Bacteria transfer DNA by one of three.
  1. Transformation – DNA is taken from fluid surrounding the cell into int and it is incorporated into the bacterial DNA.
  2. Transduction – A piece of DNA from the phage's previous host cell has been accidentally packaged inside the phage's coat. When this phage attaches to new bacterium, “stowaway” DNA is incorporated into host's DNA.
  3. Conjugation – Donor cell uses sex pilli to find recipient. They approximate each other to build a cytoplasmic bridge and “male” donor gives replicated piece of its DNA to recipient to be incorporated into its DNA.

Bacteria that can initiate conjugation contain a piece of DNA called the F factor incorporated into its DNA. This has origin of replication , and the F factor and some other DA pass to recipient cell.

If F factor is not part of the host cell's DNA strand but instead exists separately, it is called a plasmid. When this is transferred it may take bacterial DNA also.

R plasmids carry genes for making proteins that destroy antibotics.

Plasmids are often used as vectors to transfer DNA fragments from one cell (or organism) to another.

Desired DNA fragments can be “cut” from a chromosome in one cell and “pasted”into DNA of another cell using restriction enzymes and plasmids.

Restriction enzymes in organisms prevent intrusion of foreign DNA from viruses or phages. They chop up the foreign DNA.

Most restriction enzymes recognize short DNA sequences and cut at specific points within these sequences.

The staggered cuts by restriction enzymes produced DNA fragments with single stranded ends called “sticky ends”. These “sticky ends” can then form hydrogen bonds with “sticky ends” produced when the same restriction enzyme was used on a different DNA source. The bonds can be solidified by DNA ligase which helps form covalent bonds between adjacent nucleotides. The final molecule is recombinant DNA. The recombinant DNA within a cell then is replicated and passed on to daughter cells. The spliced in gene then causes all daughter cells to make the desired protein.

In gel electrophoresis, DNA fragments migrate from negative to positive because phosphate group has a negative charge. The larger the fragment length of DNA, the less distinctive it will move from the negative pole.

Restriction Fragment Length Polymorphisms RFLPs (Rif Lips):
The differences in DNA sequences on homologus chromosomes result in sequences on homologous chromosomes result in restriction fragments of different lengths (RFLIPS)

PCR method is used to amplify DNA sequences. DNA that is not in a cell is cloned in a test tube. DNA polymerase, nucleotides, and other crucial ingredients are put in test tube. These sequences can be analyzed by RFLP to determine if present in unknown sample.

Can be spliced.

Can make base sequencing easier (reactions are amplified).

There are three billion nucleotide pairs in the human genome.

Mendelian Genetics:

Before you go any further in the study of Mendelian Genetics and Probability, you should make sure that you are familiar with the following terms.

Gene - a discrete unit of heredity information consisting of a specific nucleotide sequence in DNA (or RNA in some viruses). Most of the genes of a eukaryote are located in its chromosomal DNA.

Locus - The particular site where a gene is found on a chromosome. Homologous chromosomes have corresponding gene loci.

Allele - An alternate version of a gene.

Dominant allele - The allele that determines the phenotype of a gene when the individual is heterozygous for that gene.

Recessive allele - An allele that has no noticeable affect on the phenotype of a gene when the individual is heterozygous for that gene.

Genotype - The genetic make up of an organism.

Phenotype - The expressed traits of an organism.

Homologous chromosomes - The two chromosomes that make up a mated pair in a diploid cell.

Diploid - An organism that reproduces sexually, contains two homozygous sets of chromosomes (one from each parent).

Haploid - An organism that reproduces asexually and contains a single set of chromosomes.

Heterozygous - Having two alleles for a given gene.

Homozygous - Having two identical alleles for a given gene.
In the study of genetics, the term probability is the likelihood of an event happening. An event certain to occur has a probability of 1. An event that is certain not to occur has a probability of 0.

- Chance has no memory.

Compound events are the product of the separate probabilities of an independent event.

In incomplete dominance, both alleles for the trait have an effect on the outcome. In codominance, both alleles are fully expressed in heterozygous individuals.If there is multiple allies, each individual carries, at most, two different alleles per trait, but no more than two exist.

In polygenetic inheritance, the additive effects of two or more genes on a single phenotype characteristic. Most of these characteristics vary along a continuum. They exhibit incomplete dominance. Each gene is inherited separately. Because the genes have an additive effect, the genotype AaBbCc (3 dominant and 3 recessive) produces the same skin color as AABbcc (3 dominant and 3 recessive).

Linked genes appear on the same chromosome. The closer together the loci of the genes are on the chromosomes, the more likely it is that they will be inherited together (not separated during crossing over).

Sex-linked genes are unrelated to sex determination, they are most often found on the X chromosome. The Y chromosome is much smaller than the X and carries less information because of this, males have only the information on the X chromosome from their mothers for some traits.If that gene carries a recessive "faulty" gene there is no second gene trait so it appears.

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