This is old Lecture 20

Text Assignment: Chapter 10, pages 262-297

Expected level of understanding: This is the first of a series of four lectures that provide a quick review of basic molecular biology for the benefit of members of the class who may not have taken MCDB 1150 or another course that covers the material in similar depth. The Fall 1998 syllabus for MCDB 1150 devotes at least 9 lectures directly to various aspects of DNA, RNA and proteins. The quick reviews presented in this lecture and the three that follow on DNA replication, transcription and translation will summarize the material and provide textbook references for more detailed presentation, but they are not comparable to the depth of coverage in MCDB 1150. Since MCDB 3120 and MCDB 3500 will assume that you already have the background provided by MCDB 1150 (and this course), it is important for all MCDB majors who did not take MCDB 1150 to make an added effort to learn thoroughly any parts of this material that you may not already know well. Please be sure that you are familiar with all of the terms and concepts in the lecture outline, even though we will not have enough time to cover all of them fully in class.

MCDB 1150 second examination: For a better view of the level of understanding of these materials that is expected in MCDB 1150, you may want to look at the answer key to their second examination, which should be posted in the glass case opposite Porter B113 within the next few days (the examination was given on October 12).

"Central dogma" of molecular biology:

• Genetic information is stored as a sequence of nucleotide bases (adenine, cytosine, guanine, thymine, abbreviated A,C,G,T) read sequentially in a 5' to 3' direction in DNA (or in RNA, with uracil, abbreviated U, replacing thymine).

• The most common form of DNA (present in all cellular genomes, as well as many viral genomes) is double stranded. The 5' to 3' polarity of the two strands is opposite, and they are held together by hydrogen bonding between nucleotide base pairs, A to T and G to C. The sense strand carries the coded genetic information. The antisense strand consists of a complementary sequence of bases oriented in the opposite 5' to 3' direction.

• During DNA replication, the two strands separate and each is used as a template for synthesis of a new complementary strand. This allows genetic information to be replicated with a high level of precision. Because replication is bidirectional, but new DNA can only be synthesized in a 5' to 3' direction, the overall pattern of replication is rather complex as will be discussed in the next lecture.

• Genetic information is transcribed from DNA to RNA, with the antisense strand of the DNA serving as a template for synthesis of an RNA with the same base sequence (5' to 3') as the sense strand of the double helical DNA, except that uracil (U) replaces thymine (T).

• Genetic information contained in messenger RNA (mRNA) is translated into a sequence of amino acids in a polypeptide chain during protein synthesis (translation). A redundant nucleotide triplet code, read 5' to 3' on the mRNA (and on the sense strand of the DNA), specifies the amino acid sequence of the protein, read from N-terminal to C-terminal.

Prokaryotic and eukaryotic cells

• There are two basic types of cells, called eukaryotic and prokaryotic respectively (sometimes spelled eucaryotic and procaryotic).

• Eukaryotic cells have their genetic material separated from the cytoplasm in a membrane-bound nucleus. The nuclear envelope restricts and regulates the passage of materials between the two compartments. DNA synthesis and transcription (RNA synthesis) occur within the nucleus. Messenger RNA (mRNA) is then processed and transported to the cytoplasm for use as a template for translation (protein synthesis). This causes translation to be separated both in space and in time from transcription.

• Prokaryotic cells, which include bacteria and some primitive algae, are much smaller and have a simpler structure, with no true separation of DNA from the cytoplasm. Because of this, transcription and translation occur in the same space and overlap in time. In fact, translation of an mRNA will normally begin soon after its transcription has been initiated, and long before synthesis of a full-length message has been completed.

Note: Because of the large amount of text material covered and the review nature of the material, the following sections are presented only as outlines.

DNA and RNA as carriers of genetic information

• Double stranded DNA is the genetic material in all eukaryotic and prokaryotic cells, as well as mitochondria, chloroplasts, plasmids and many types of viruses..

• Some types of viruses have single stranded DNA genomes.

• Some types of viruses have single stranded or double stranded RNA genomes

Evidence for role of nucleic acids as carriers of genetic informaiton

• Avery, McLeod, & McCarty: Genetic transformation of bacteria with DNA

• Hershey-Chase Experiment: Labeled bacteriophage DNA enters cells during infection but labeled bacteriophage protein does not.

• Transfection of bacterial spheroplasts with bacteriophage DNA.

• Recombinant DNA technology, production of transgenic mice

Chemistry of nucleic acid components

• Pentose sugars (Ribose in RNA, Deoxyribose in DNA).
• Sugar phosphate esters.
• Phosphodiester bonds (3' to 5' bonding in nucleic acid backbones).
• 5' and 3' ends of polynucleotides, 5' to 3' directionality
• Purine and pyrimidine bases attach to 1' positions of ribose and deoxyribose
• Purine bases (Adenine, Guanine).
• Pyrimidine bases (Cytosine, Uracil in RNA, Thymine in DNA).
• Nucleosides (base attached to ribose): adenosine, guanosine, cytidine, uridine.
• Deoxynucleosides (base attached to deoxyribose): deoxyadenosine, deosyguanosine, deoxycytidine, thymidine.
• Nucleotides (base attached to ribose phosphate): AMP, GMP, CMP, UMP.
• deoxynucleotides (base attached to deoxyribose phosphate): dAMP, dGMP, dCMP, TMP (or dTMP)
• Nucleotide triphosphates (base attached to ribose 5'-triphosphate): ATP, GTP, CTP, UTP.
• Deoxynucleotide triphosphates (base attached to deoxyribose 5'-triphosphate): dATP, dGTP, dCTP, TTP (or dTTP)

Structural features of double helical DNA

• Nucleotide sequences are normally read 5' to 3'.
• Nucleotide base pairing, AT and GC pairs.
• Double helix, antiparallel orientation, stacked base pairs.
• Separation of DNA strands (melting) occurs at high salt levels and/or high temperature.
• GC base pairs have 3 hydrogen bonds and are more stable than AT base pairs, which only have 2.
• Stability of double-helical DNA increases with higher GC content.
• Separated strands can reform a double helix (anneal) if denaturating conditions are slowly reversed.
• The amount of time required to renature increases with the compexity of the mixture of sequences present.
• Complexity of DNA (total number of nucleotides of unique sequence) is often measured in terms of the product of total nucleotide concentration and time required for 50% renaturation (C_ot values)
• Complex genomes, such as those of mammals, contain a mixture of repetitive and unique sequence DNA.
• In addition to the common B configuration, double stranded DNA can also assume other configurations, some of which may be biologically significant.

• RNA is much like a single strand of DNA, and can pair with a single complementary strand of DNA to form an antiparallel double helical structure.
• RNA molecules may fold back on themselves and form limited double-stranded regions, which appear to be important for achieving active configurations.
• Three major classes of RNA are involved in protien synthesis, messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA).
• RNA sequences are read 5' to 3' in the same manner as DNA.
• The coding sequence in mRNA is the same as that of the sense strand of DNA, except that U replaces T.
• Small nuclear RNA (snRNA) molecules have a variety of biological functions.

Electrophoresis

• Nucleic acids have strong negative charges at neutral pH, and thus move toward the annode (positive pole) in an electric field.
• The rate of movement through a porous gel (polyacrylamide or agarose) is faster for smaller molecules and slower for larger molecules.
• Gel electrophoresis is a highly effective technique for separating mixtures of nucleic acids by size (Figure 10.27)
• Electrophoretic separations play major roles in many of the molecular techniques we will be studying.

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