Samford Howard College of Arts and Sciences Logo
Department of Biological and Environmental Sciences

Cell & Molecular Biology
Dr. David A. Johnson
Biol 405    4 Credits   Spring 2017  MWF 11:45-12:50 AM   PH
204

A Brief Overview of Cells and Cell Biology   (ch.1 pp. 1-12, 17-38)

Living things are composed of cells. Cells of archaea (domain: archaea) and bacteria (domain: bacteria) are quite different from those of the rest of the living world. They make up the prokaryotes, while the rest (from protozoa to you) are eukaryotes (domain: eukarya).
ecoli
E. coli

Prokaryotes: Prokaryotes include the archaea (many inhabiting harsh environments) and bacteria (including the photosynthetic cyanobacteria). They lack a nucleus (no nuclear membrane) but have a region called a nucleoid. They have ribosomes but lack other cellular organelles. The prokaryote chromosome is a "naked" circular DNA molecule. As we will see in detail later, they also lack the process of splicing.

prok

Eukaryotes: Eukaryotes have a double nuclear membrane separating the chromosomes from the rest of the cell. (Plant cell, animal cell) Their chromosomes are linear DNA molecules bound to protein (the most abundant protein being histones). The process of splicing is seen only in eukaryotes. In addition to ribosomes, they have a variety of organelles and structures including these and others (details later):

Animal Cell

Plant Cell

  • Mitochondria (oxidative cellular respiration)
  • Chloroplasts (photosynthesis)(found in plant and algal cells)
  • Lysosomes
  • Peroxisomes
  • Endoplasmic reticulum (continuous with the outer nuclear membrane)(cytosol, lumen of the ER)
  • Golgi apparatus (secretion)
  • Cytoskeleton
Miller bilayer

Origin of Cells: The first cell are thought to have arisen from the pre-biotic mixture in the oceans. Miller has proposed how this early chemical evolution may have occurred. Phospholipid molecules will self-assemble into a bilayer. RNA has recently been show to be able to catalyze its own replication, leading to the hypothesis of an early, RNA world (with the more stable DNA later replacing RNA as the genetic material of most organisms)(an alternative view to the RNA world - also pasted below)(Still another alternative view of the origin of life on earth!!!)(Newer evidence for the RNA world.)

 mitochondria and chloroplasts

Origin of Eukaryotes: Eukaryotes apparently arose after endosymbiosis involving a bacterium capable of oxidative phosphorylation (becoming mitochondria) and endosymbiosis of a cyanobacterium capable of photosynthesis (becoming chloroplasts). Evidence for this theory includes the structure of the DNA and the ribosomes of mitochondria.

Experimental Cell Models: Several model system have proven useful in understanding cellular processes including these:

E. coli
  • Yeast (Saccharomyces cerevisiae): This eukaryote is almost as easy to culture as bacteria. Yeast has 16 linear chromosomes composed of about 12 million bp and 6000 genes.
  • Caenorabditis: A roundworm, also easily cultured.
Coenorhabditi
Drosophila
Aribidopsis
Xenopus Zebra Fish
Microscopy: There are at least two major types of microscopes.
  • Light Microscope: Magnification and resolution are both important functions of microscopes. The limits of light microscope magnification are around 1000x (due to the limit on the resolving power of light, resolution is dependent on the wavelength--about 500 nm for green light). Light microscopes use glass lenses and include bright field, phase contrast, and fluorescence microscopes (microtubles fluoresce green below).
light
                  microscope 

Bright Field stained phase contrast microscope

 Fluorescent Microscope Fluorescent Microscopy
  • Electron Microscopes: The electron microscope uses a beam of electrons and therefore has greater resolving power (wavelength < 0.01 nm). Magnification of up to 1,000,000x are possible with the transmission (versus scanning) electron microscope. The lenses of an EM are magnets, which can bend a beam of electrons.
Transmission EM photo Scanning EM photo
Fractionation and Separation of Cell Components: Cell components can be separated by several methods.
  • Differential Centrifugation: This techniques uses multiple rounds of centrifugation at increasingly higher g forces. After each round, the supernatant is removed and centrifuged in the next round.
Differential
                Centrifugation
  • Gradient Centrifugation is of two overall types:
    • Velocity Centrifugation (Sucrose Gradient Centrifugation): Molecules or particles are sedimented through a gradient (increasing concentration at the bottom of the tube). Molecules with a higher molecular weight sediment faster. This procedure does NOT go to equilibrium. This method separates molecules according to sedimentation velocity, S (Svedberg units).
Sucrose Gradient
    • Equilibrium Centrifugation (CsCl Density-Gradient Centrifugation) sediments molecules through a density gradient and separates molecules according to density. (Goes to equilibrium.)(CsCl centrifugation video)

Cell Culture: Primary and secondary cultures of human cells will eventually stop dividing. Stem cell line cultures (as well as cancer cell lines) keep dividing. (January, 2012 news: Using embryonic stem cells to treat macular degeneration?)(The book "The Immortal Life of Henrietta Lacks" Published by Random House is available in paperback.)

Units Used to Measure Cells

units  

 

(The material below will not be on any exam.)
Something to Think About

(Stolen from Dr. Daniel P. Heruth, former chair, Biology Department, William Jewell College, Liberty, MO)

The process of life is played out within the cell. Therefore an understanding of the dimensions of the cell and its contents is important to the study of cell and molecular biology. It is important to realize that the cell is a very crowded place, and thus very organized. A typical eukaryotic cell has a diameter of 25 μm. If we magnified the cell by a million fold, we would have a cell 25M in diameter. Twenty-five meters is approximately 81 feet , so if we were standing in the center of the cell it would be 40 feet in every direction. Our magnified cell would be approximately the size of a large college lecture room, if we removed the seating. A water molecule, the most common molecule within a cell, has a diameter of 0.4 nm. Magnified a million-fold, the water molecule would be only 0.4 mm in diameter. This is the size of the period at the end of this sentence. Amino acids, the building blocks of proteins are larger than water molecules. They have an average molecular weight of 110 and are from 0.6 nm - 1.2 nm in length. Increased by a factor of a million, the amino acids would be 0.6 mm - 1.2 mm in length or about three times larger than the period. Even at this scale, we would not be able to distinguish the atoms within the amino acid. Hemoglobin, a protein made from amino acids has a molecular weight of 64,000 and a diameter of 6.4 nm. Magnified it would have a diameter of 6.4 mm or the size of a pea. Ribosomes, large complexes of proteins and RNA, has a diameter of 25 nm. Magnified a million-fold would increase the diameter to 25 mm, the size of a ping pong ball. In a typical eukaryotic cell there are more than 20,000 ribosomes. A mitochondrial organelle, the site of ATP synthesis, is 1μm in length; amplified it would become a 1 m long oversized football, much like a rugby ball. A typical liver cell, a hepatocyte contains 800-2500 mitochondria (~20% cell volume). A chloroplast is 5X larger than a mitochondrion, so if our cell was a plant cell you can imagine that we may be running out of room. DNA is 2 nm wide, so in our cell it would be a 2 mm wide strand of sewing thread. A human cell contains 1.8 m of DNA. Therefore, if we were to stretch out all of the DNA in our magnified cell end to end we could extend it over 19,500 football fields. Remarkably, this DNA would need to be coiled around histone proteins and packaged tightly within the nucleus, a membrane bound region found in the center of our cell. This is possible because DNA is a very thin molecule. Even more remarkable is the plasma membrane which surrounds and protects the cell. Magnified a million-fold, the membrane would be only 1 cm thick. How can such a thin membrane complex of proteins and lipids define the fundamental unit of life? Welcome to the world of the cell!

New Study Contradicts RNA World Theory
03/12/2012 Tanya Lewis

XTRA--A new study proposes that proteins evolved much earlier than previously suspected, casting doubt on the hypothesis of ribosomal origins. Since the 1970s, biologists have believed that ribosomes--the molecular machinery responsible for protein synthesis--evolved from primitive RNA structures. In this RNA world hypothesis, proteins and DNA developed after the ribosome's RNA components. But now, a new paper published March 12 in PLoS ONE by scientists at the University of Illinois and Lund University in Sweden is challenging this view. In the study, researchers found evidence that proteins co-evolved with each ribosomal subunit as the modern ribosome developed. "This challenges a widely held view that the origin of the ribosome was the innovation that started to make proteins," said senior author Gustavo Caetano-Anolles. Contrary to the hypothesis that the ribosome began its development at the site known as the peptidyl transferase center (PTC) on the large ribosomal subunit, the new research found that ancient proteins were interacting with the small ribosomal subunit much earlier. In the study, the team applied phylogenetic methods to trace the evolution of the ribosome, using two approaches: one focused on RNA and the other on proteins. In the RNA approach, using data from the European Ribosomal RNA database, Caetano-Anolles and colleagues constructed phylogenetic trees by aligning ribosomal structures with molecules in the superkingdoms Archaea, Bacteria, and Eukarya. In the protein approach, the scientists made a census of all protein structures in about 750 sequenced genomes--from Escherichia coli to human--using algorithms to match known structures to sequences. Then they used that data to generate phylogenomic trees. Together, the phylogenetic and phylogenomic trees allowed them to date the addition of each molecular structure and color 2D and 3D models of the ribosome based on age. "As the RNA was [evolving], the RNA proteins were added to the ensemble at the same time," said Caetano-Anolles. "It's a scenario of gradual accretion in a world that is very much like the one of today that has nucleic acids and proteins." Although molecular biologist Russell Doolittle of the University of California, San Diego, who was not involved in the study, praised the work, he also expressed curiosity about how early proteins could be synthesized before the ribosome existed. One possible explanation is nonribosomal protein biosynthetic machinery that can make small peptides without RNA, albeit less efficiently than ribosomes. The idea that proteins preceded ribosomes is not entirely new. Nobel Laureate biochemist Fritz Lipmann proposed the idea in the 1970s, but the idea was overshadowed by the RNA World theory, which was fueled by the synthesis of the first ribozymes--enzymes comprised of RNA--in the laboratory."We have to rethink completely our point of view about the origin of life," said Caetano-Anolles. "It's not an RNA world--it's gradual and messy."

BUT: Some even newer evidence says maybe the RNA world idea is right after all:

Shedding Light on the Origins of Life
BIOTECHNIQUES
10/17/2016 Janelle Weaver, PhD

According to the RNA world hypothesis, self-replicating RNA molecules were the precursors to all current life on Earth. Ancestral organisms contained RNA genes that were replicated by RNA enzymes, and it was only later that genetic information and catalytic functions were transferred to DNA and proteins, respectively.
This idea has gained traction over the past several decades and is now widely accepted. Major support for this hypothesis came in the early 1980s with the discovery that RNA is not only a molecule of heredity but that it could also serve as an enzyme. This seminal research earned Sidney Altman and Thomas Cech the Nobel Prize in Chemistry in 1989, and since then, many scientists have focused on reconstructing RNA-based life in the lab.

One of these scientists is David Horning, a research associate in the lab of Gerald Joyce at the Scripps Research Institute. His group is on a mission to understand how life emerged on Earth and possibly other planets.
"In particular, we are trying to understand how extremely simple life made only of RNA, which is believed to have preceded all modern life, could grow and evolve," Horning said. "Since RNA life probably went extinct billions of years ago, we need to build RNA life from scratch in the lab, and to do that, we need an RNA enzyme that can replicate other RNAs, including itself."

Horning and Joyce moved closer to that goal in a recent study published in Proceedings of the National Academy of Sciences (PNAS) [1]. They reported the in vitro evolution of an improved RNA polymerase ribozyme that can synthesize structured functional RNAs and replicate short RNA sequences in a protein-free form of PCR. According to the authors, the new findings demonstrate that the two prerequisites of Darwinian life--replication of genetic information and its conversion into functional molecules--can now be accomplished with RNA in the complete absence of proteins.

"We see this result as an important milestone on the way to a much broader goal: constructing a chemical system that is able to replicate itself and evolve," Horning said. "We think this step brings us much closer, and now the focus is on improving the replication activity of the polymerase to get to the point where it's able to replicate itself or other ribozymes of similar complexity.
"
Reconstructing RNA-Based Life:
In the new study, Horning and Joyce used in vitro evolution to dramatically improve the activity and generality of an RNA polymerase ribozyme by selecting variants that could synthesize functional RNA molecules from an RNA template. The improved polymerase ribozyme, isolated after 24 rounds of evolution, synthesized a variety of structurally complex RNAs, including aptamers, ribozymes, and even tRNAs. Moreover, the polymerase replicated nucleic acids, amplifying short RNA templates by more than 10,000-fold in an RNA-catalyzed form of PCR. According to the authors, the improved polymerase, designated 24-3, is the first known ribozyme that can amplify RNA and synthesize complex functional RNAs.

This research builds on previous efforts in the Joyce lab. In a study published in Science in 2009, the researchers reported cross-replicating RNA enzymes that underwent self-sustained exponential amplification in the absence of proteins or other biological materials [2]. And in a study published 5 years later in Nature, the lab used in vitro evolution to develop a cross-chiral RNA polymerase, supporting the notion of a primordial RNA world in which cycles of cross-handed replication used mirror-image forms of RNA [3].

"The former [study] did make a set of ribozymes that were mutually self replicating, but they were highly specific to replicating particular RNA structures and could not generally replicate most RNA," Horning explained. "The latter built an RNA polymerase ribozyme that could synthesize RNA of the opposite handedness (L-RNA). At the time, it was the most active polymerase ribozyme discovered, although our unrelated 24-3 has now surpassed it."

According to Horning, this research could have a variety of practical applications. "First, we are able to amplify RNA by PCR directly, rather than converting it into DNA, which could be useful in studying or modifying biological RNA," Horning said. "The polymerase may also be tweaked to accept non-natural nucleic acids, which might allow us to more readily use a variety of more arcane nucleic acid variants which have proven useful in biotechnology."
 
Evolutionary Implications:
Beyond its practical applications, the new approach could shed light on the early evolution of life.
"This paper seems like a big deal to me," said Sean Eddy, who studies the evolutionary history of life at Harvard University and was not involved in the research. "As far as I know, this is the first time that someone has demonstrated a ribo-polymerase capable of copying essentially any RNA template from standard nucleotides. This is a major success and a big step forward toward showing that RNA can replicate itself."
 
But according to Eddy, there are still
"plenty of issues remaining in achieving a plausible path to this sort of system in the prebiotic world." Other experts agree. For example, Ulrich Muller, who studies the evolution of catalytic RNAs at the University of California, San Diego, pointed out that "the current system does not have an efficient method for strand displacement during PCR amplification by the polymerase ribozyme." Another potential drawback is that "the procedure requires thermocycling, just like conventional DNA-based PCR," noted Peter Unrau, an expert on RNA chemistry and evolution at Simon Fraser University who served as one of the reviewers of the PNAS paper.

For his own part, Horning conceded that the polymerase is not yet active enough to make a new copy of itself or replicate most longer RNA sequences.
"We think more directed evolution should be able to move past these limitations," he said. "If our lab or another gets to that point, it should be possible to construct a simple system where the polymerase can sustain its own replication indefinitely and potentially continue to grow and evolve as its own, independent life form."
 
References

1. Horning, DP, Joyce GF (2016) Amplification of RNA by an RNA polymerase ribozyme. PNAS 113(35): 9786-9791. DOI: 10.1073/pnas.1610103113.
2. Lincoln, TA & Joyce, GF (2009) Self-sustained replication of an RNA enzyme. Science 323(5918):1229-1232. DOI: 10.1126/science.1167856.
3. Sczepanski JT, Joyce GF (2014) A cross-chiral RNA polymerase ribozyme. Nature 515(7527):440-2. DOI: 10.1038/nature13900..

Pun of the Day: I know a guy who's addicted to brake fluid. He says he can stop anytime.