We're currently fixing things up at the moment.
Meanwhile, here's an article on how why light eyes may be more attractive. Limbal rings on light eyes are more noticeable, by the way.
Saturday, September 17, 2011
Wednesday, September 14, 2011
TTAG Summary: The transcriptional program of sporulation in budding yeast.
S. Chu, J. DeRisi, M. Eisen, J. Mulholland, D. Botstein, P. O. Brown, I. Herskowitz. 1998. The Transcriptional Program of Sporulation in Budding Yeast. Science, 282, 699-705.
Science 1998 Nov 20;282(5393):1421.
Here's my review on the paper.
So under starvation conditions, diploid Saccharomyces cerevisiae produce stress-resistant haploid spores in a process called sporulation. In order to successfully sporulate, diploid cells must undergo meiosis to reduce the ploidy of cells, and spore morphogenesis to package the genome in a prospore membrane, then the protective spore wall. The complexity of sporulation requires regulated changes in gene expression in order for the stages to progress in a coordinated, sequential manner.
The authors of the article are investigating every gene involved in sporulation and how different regulatory sites will affect when these genes are induced. The authors intend to categorize these genes based on their time of induction then link each category’s unique temporal profile to changes within the cell during the course of sporulation. Thus, they are able to hypothesize the functions of genes with previously unknown functions based on the time of their induction during sporulation.
To induce sporulation, cells were put on nitrogen-deficient medium. Sporulation is initiated by deficiencies in nitrogen and fermentable carbon sources, inducing a signal transduction pathway and arresting the cell at G1. At 7 different time points after sporulation initiation, they did a microarray analysis of 97% of all yeast genes to measure the changing abundance of mRNA transcripts of each gene over time compared to vegetative cells. The 7 different time points were based on the time of induction of DMC1, SPS1, DIT1, and SPS100, which represent the expression patterns for early, middle, mid-late, and late genes, respectively. Northern and microarray analysis were used to confirm the time of induction and relative transcript abundance of each of the four genes. Results from the microarray showed that, over the course of sporulation, 500 genes were induced and about 500 were repressed. The induced genes were categorized into seven groups (Metabolic, Early I, Early II, Early Middle, Middle, Late-Middle, and Late) based on the time of initial induction.
The metabolic group is induced first, and functions to acclimatize the cells to nitrogen-starved conditions. They have different temporal expression profiles, despite having the same URS1 sequence, and so must be regulated by other means. The majority of genes with the early I pattern have a URS1 site or a core URS1 site. They are induced after .5 hours and play a role in synapsis and homologous recombination. Genes with the early II pattern are less likely than early I genes to have putative URS1 sites, which may explain why their induction is slightly delayed. Early-middle genes are induced at 2 hours and continue to increase even after7 hours. Some genes that display this pattern have a role in spindle pole body dynamics. Middle genes, induced from 2-5 hours, are regulated primarily by Ndt80. Ectopic expression induced many genes with an MSE site in vegetative cells. Genes in vegetative cells that remain uninduced are likely to require additional factors. In addition to expressing Ndt80 in non-sporulating cells, the authors inactivated Ndt80 expression in sporulating cells. Many genes were expressed at a third the level of wild-type cells, resulting in arrest during prophase. However, other genes, induced independently of Ndt80, required additional input to be induced. The mid-late class of genes is induced after 5-7 hours, and the genes usually have an NRE (negative regulatory element) site in addition to the MSE site that delays their induction compared to middle genes. This class includes genes that contribute to prospore membrane formation. The late class of genes is induced after 7 hours and includes genes involved in spore maturation. Each class of genes has a common regulatory sequence in the promoter region targeted by a specific transcription factor. Binding of the transcription factor to its target site induces the gene’s transcription. Ume6/Ime1 recognizes the URS1 (Ume cognate cis-acting regulatory sequence) found upstream of early genes. Genes with MSE sites (middle gene sporulation element) induced by Ndt80 protein. Proteins that induce mid-late and late genes have not been discovered at the time of publication. Temporal expression patterns during sporulation are possible due to sequences such as URS1 and MSE, allowing induction of an entire set of genes at different stages of meiosis and spore morphogenesis when they are required. By linking what a gene does to how it is regulated by different transcription factors, the authors are able describe the general program of sporulation.
They needed to show that gene induction of the different classes was happened concurrently with physiological changes in cell, so the cells were assessed cytologically. At various time points, DAPI (4’-6’diaminidino-2-phenylindole) was used to stain the nuclei of sporulating cells to determine how many nuclei were present in the cells and measure the rate of meiosis. At around 5 hours, after the middle genes have been induced, mononucleate cells become binucleate. By 9 hours, tetranucleate cells outnumber mononucleate cells. Electron microscopy was used to determine the progress of spore maturation. From 5 to 9 hours, the rate of spore formation rose dramatically: from 0 percent to under 40. By 11.5 hours, the percent of mature spores had risen to equal that of immature spores. These results support the role of mid-late genes in prospore membrane formation and late genes in spore maturation because the cytological changes coincided with the gene’s induction.
The authors’ results show that in sporulation the time of induction of a gene is strongly correlated to its function and the mechanism of regulation. This correlation makes genes with unknown functions candidates for different processes based on their time of induction. The authors use this correlation as part of their strategy to propose the roles many genes such as Spo69, Spo70, and Spo71. The authors also compare homologous proteins with known functions in Drosophila and Xenopus and Caenorhabditis elegans to Spo70, a sporulation-specific protein, to determine whether the protein had the same function in yeast. In conclusion, the authors used temporal induction patterns of every gene in S. cerevisiae to propose gene functions for many previously uncharacterized genes. Genes each class had characteristic regulatory sequences that determined the time of induction. Time of induction during sporulation is tied with gene function as transcripts are induced when they have a role to play in the specific stages of the program. Thus, regulatory sites such as MSE and URS1 can be used to determine gene function.
Science 1998 Nov 20;282(5393):1421.
Here's my review on the paper.
So under starvation conditions, diploid Saccharomyces cerevisiae produce stress-resistant haploid spores in a process called sporulation. In order to successfully sporulate, diploid cells must undergo meiosis to reduce the ploidy of cells, and spore morphogenesis to package the genome in a prospore membrane, then the protective spore wall. The complexity of sporulation requires regulated changes in gene expression in order for the stages to progress in a coordinated, sequential manner.
The authors of the article are investigating every gene involved in sporulation and how different regulatory sites will affect when these genes are induced. The authors intend to categorize these genes based on their time of induction then link each category’s unique temporal profile to changes within the cell during the course of sporulation. Thus, they are able to hypothesize the functions of genes with previously unknown functions based on the time of their induction during sporulation.
To induce sporulation, cells were put on nitrogen-deficient medium. Sporulation is initiated by deficiencies in nitrogen and fermentable carbon sources, inducing a signal transduction pathway and arresting the cell at G1. At 7 different time points after sporulation initiation, they did a microarray analysis of 97% of all yeast genes to measure the changing abundance of mRNA transcripts of each gene over time compared to vegetative cells. The 7 different time points were based on the time of induction of DMC1, SPS1, DIT1, and SPS100, which represent the expression patterns for early, middle, mid-late, and late genes, respectively. Northern and microarray analysis were used to confirm the time of induction and relative transcript abundance of each of the four genes. Results from the microarray showed that, over the course of sporulation, 500 genes were induced and about 500 were repressed. The induced genes were categorized into seven groups (Metabolic, Early I, Early II, Early Middle, Middle, Late-Middle, and Late) based on the time of initial induction.
The metabolic group is induced first, and functions to acclimatize the cells to nitrogen-starved conditions. They have different temporal expression profiles, despite having the same URS1 sequence, and so must be regulated by other means. The majority of genes with the early I pattern have a URS1 site or a core URS1 site. They are induced after .5 hours and play a role in synapsis and homologous recombination. Genes with the early II pattern are less likely than early I genes to have putative URS1 sites, which may explain why their induction is slightly delayed. Early-middle genes are induced at 2 hours and continue to increase even after7 hours. Some genes that display this pattern have a role in spindle pole body dynamics. Middle genes, induced from 2-5 hours, are regulated primarily by Ndt80. Ectopic expression induced many genes with an MSE site in vegetative cells. Genes in vegetative cells that remain uninduced are likely to require additional factors. In addition to expressing Ndt80 in non-sporulating cells, the authors inactivated Ndt80 expression in sporulating cells. Many genes were expressed at a third the level of wild-type cells, resulting in arrest during prophase. However, other genes, induced independently of Ndt80, required additional input to be induced. The mid-late class of genes is induced after 5-7 hours, and the genes usually have an NRE (negative regulatory element) site in addition to the MSE site that delays their induction compared to middle genes. This class includes genes that contribute to prospore membrane formation. The late class of genes is induced after 7 hours and includes genes involved in spore maturation. Each class of genes has a common regulatory sequence in the promoter region targeted by a specific transcription factor. Binding of the transcription factor to its target site induces the gene’s transcription. Ume6/Ime1 recognizes the URS1 (Ume cognate cis-acting regulatory sequence) found upstream of early genes. Genes with MSE sites (middle gene sporulation element) induced by Ndt80 protein. Proteins that induce mid-late and late genes have not been discovered at the time of publication. Temporal expression patterns during sporulation are possible due to sequences such as URS1 and MSE, allowing induction of an entire set of genes at different stages of meiosis and spore morphogenesis when they are required. By linking what a gene does to how it is regulated by different transcription factors, the authors are able describe the general program of sporulation.
They needed to show that gene induction of the different classes was happened concurrently with physiological changes in cell, so the cells were assessed cytologically. At various time points, DAPI (4’-6’diaminidino-2-phenylindole) was used to stain the nuclei of sporulating cells to determine how many nuclei were present in the cells and measure the rate of meiosis. At around 5 hours, after the middle genes have been induced, mononucleate cells become binucleate. By 9 hours, tetranucleate cells outnumber mononucleate cells. Electron microscopy was used to determine the progress of spore maturation. From 5 to 9 hours, the rate of spore formation rose dramatically: from 0 percent to under 40. By 11.5 hours, the percent of mature spores had risen to equal that of immature spores. These results support the role of mid-late genes in prospore membrane formation and late genes in spore maturation because the cytological changes coincided with the gene’s induction.
The authors’ results show that in sporulation the time of induction of a gene is strongly correlated to its function and the mechanism of regulation. This correlation makes genes with unknown functions candidates for different processes based on their time of induction. The authors use this correlation as part of their strategy to propose the roles many genes such as Spo69, Spo70, and Spo71. The authors also compare homologous proteins with known functions in Drosophila and Xenopus and Caenorhabditis elegans to Spo70, a sporulation-specific protein, to determine whether the protein had the same function in yeast. In conclusion, the authors used temporal induction patterns of every gene in S. cerevisiae to propose gene functions for many previously uncharacterized genes. Genes each class had characteristic regulatory sequences that determined the time of induction. Time of induction during sporulation is tied with gene function as transcripts are induced when they have a role to play in the specific stages of the program. Thus, regulatory sites such as MSE and URS1 can be used to determine gene function.
Tuesday, September 13, 2011
Question of the Day: Liquid Mechanics Involving Protein Powder
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| WHY DOESN'T THIS EVER MIX WELL IN WATER?!?! |
Headache01 asks "Why doesn't powder mixed into drinks (like whey protein powders or cocoa) dissolve very well? It clumps into balls that are wet on the outside but remain dry on the inside. How can I make my whey mix better?"
Well, given that proteins, and especially mixture of proteins, are amphiphilic (meaning the molecule has a part with an affinity for water, and another for fat/oils), they tend to orient themselves into microscopic structures known as micelles. Micelles allow the molecules to isolate their lipophilic ends away from the water and their hydrophilic end towards water, ultimately forming a spherical, tubular, or sheetlike structure.
While micelles are a microscopic phenomena, a similar thing is what causes the clumps in your protein drink; along the protein powder-water interface there will be two-layer sheet type structures where the hydrophilic parts of the molecules are facing the water and the lipophilic parts stay away from it.
Wonder why you should avoid warm water when mixing powders? The heat causes the proteins to lose their secondary structure and become entangled with one another, making it difficult to break up the clump since the clump's outer surface has essentially polymerized. Thus, using cold water keeps the proteins tightly coiled and less likely to get entangled with each other.
I almost forgot! The solution is to wet each of the solid particles individually first before dispersing them (e.g. mix in a small amount of water to form a paste). This will ensure that they disperse well.
While micelles are a microscopic phenomena, a similar thing is what causes the clumps in your protein drink; along the protein powder-water interface there will be two-layer sheet type structures where the hydrophilic parts of the molecules are facing the water and the lipophilic parts stay away from it.
Wonder why you should avoid warm water when mixing powders? The heat causes the proteins to lose their secondary structure and become entangled with one another, making it difficult to break up the clump since the clump's outer surface has essentially polymerized. Thus, using cold water keeps the proteins tightly coiled and less likely to get entangled with each other.
I almost forgot! The solution is to wet each of the solid particles individually first before dispersing them (e.g. mix in a small amount of water to form a paste). This will ensure that they disperse well.
Monday, September 12, 2011
DNA Repair Systems
During replication, DNA polymerase proofreads the newly synthesized strand, and improperly incorporated bases are removed by its 3' to 5’ exonuclease activity. In addition to proofreading, replication errors are corrected by the mismatch repair system. Mismatched bases change the conformation of the helix. In the mismatch repair system, the distortion is recognized and the region around the newly synthesized strand is excised. Some bacteria use methylase to differentiate between the old and newly synthesized strand. DNA polymerase then fills in the gap, using the older strand as a template. Other global systems commonly repair DNA in cells: base excision, photolyase, and nucleotide excision repair. In base excision repair, DNA glycosylase recognizes specific faulty bases, and hydrolyzes the glycosidic bond between the base and the deoxyribose backbone. AP endo/exonuclease then excises the single deoxyribose, and DNA polymerase fills in the gap. In photoreactivation, DNA photolyase recognizes and binds to thymine dimers, which cause a conformational change in the DNA helix. When light activates this enzyme, it breaks the covalent bonds of the ring, reversing UV damage. The nucleotide excision repair, helicases melt the duplex at the site of distortions, and a 12-13 residue long single-stranded DNA segment at this site is excised. DNA polymerase then fills in the gap left behind.
DNA damage can involve more than just wrongly incorporated bases. When double strand breaks or gaps occur, they are repaired either by non-homologous end joining (before replication) or homologous recombination (after replication, when sister chromatids can provide a template). In NHEJ, an exonuclease process the single stranded ends of the broken DNA, and ligases then directly join them together. The digestion of ends may result in the loss of nucleotides and mutations. Homologous recombination is less mutagenic because sister chromatids or other homologous regions are used as a template for repairing the gap.
Some mutations are induced as part of a cell’s response to stress and DNA damage. The presence of ssDNA induces the SOS response in E. coli. RecA binds to ssDNA filaments and ATP, activating RecA. Active RecA induces self-cleavage of the repressor of din genes, LexA, and the din genes, including the umuDC operon, are transcribed. The umuD and umuC gene products form a heterotrimer UmuD’(2)C, DNA Pol V, which will synthesize DNA across lesions irrespective of the residues on the template strand. For example, DNA Pol V may insert guanines opposite a thymine dimer or a cytosine opposite an apurinic site, something DNA Pol III cannot do. Thus, the SOS response and DNA Pol V allow the cell to continue replication of its genome despite DNA damage. Additionally, DNA Pol V’s mutagenic nature allows cells to mutate specifically when they are maladapted to their environment.
DNA damage can involve more than just wrongly incorporated bases. When double strand breaks or gaps occur, they are repaired either by non-homologous end joining (before replication) or homologous recombination (after replication, when sister chromatids can provide a template). In NHEJ, an exonuclease process the single stranded ends of the broken DNA, and ligases then directly join them together. The digestion of ends may result in the loss of nucleotides and mutations. Homologous recombination is less mutagenic because sister chromatids or other homologous regions are used as a template for repairing the gap.
Some mutations are induced as part of a cell’s response to stress and DNA damage. The presence of ssDNA induces the SOS response in E. coli. RecA binds to ssDNA filaments and ATP, activating RecA. Active RecA induces self-cleavage of the repressor of din genes, LexA, and the din genes, including the umuDC operon, are transcribed. The umuD and umuC gene products form a heterotrimer UmuD’(2)C, DNA Pol V, which will synthesize DNA across lesions irrespective of the residues on the template strand. For example, DNA Pol V may insert guanines opposite a thymine dimer or a cytosine opposite an apurinic site, something DNA Pol III cannot do. Thus, the SOS response and DNA Pol V allow the cell to continue replication of its genome despite DNA damage. Additionally, DNA Pol V’s mutagenic nature allows cells to mutate specifically when they are maladapted to their environment.
Sunday, September 11, 2011
Mutagens and Mutagenesis
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| Negatively charged DNA coiled around histones. Credit: Thomas Splettstoesser (from PDB 1EQZ) |
Mutations are permanent changes in the genetic material passed down from one generation to the next. In order for the continuation of the species, each generation of an organism must faithfully replicate their genomes. In order for the species to evolve and adapt, some mutations must be allowed to occur. As the substrate of evolution, mutations are especially crucial for the maintenance of genetic variation in microbial populations, which do not rely on sex or meiosis.
In the laboratory, mutagens allow geneticists to study loss-of-function mutations and elucidate gene function. For instance, the function of the gene products involved in DNA repair was elucidated by introducing mutations in their respective genes and comparing mutants’ sensitivity to chemical and ionizing mutagen to the sensitivity of the wild-type organism.
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| Nitrous acid. Don't drink it. |
For example, mitomycin C and 1,2,7,8-diepoxyoctane cause deletions while nitrous acid causes oxidative deamination of adenine and thymine to hypoxanthine and xanthine, respectively. Diepoxybutane affects G:C base pairing. N-methyl-N-nitrosourea and N-methyl-N’-nitro-N-nitrosoguanidine methylate bases, causing base-pair substitutions and baseless deoxyriboses. Ethidium bromide has a ring structure that intercalates bases and stretches the duplex, leading to frameshift mutations. Incorporation of base analogs, with higher rates of tautomerization, into DNA during replication will also lead to mutations. Ultraviolet irradiation is also mutagenic, and comes in three forms (in order of increasing energy) : UVA, UVB, UVC. UVC is the most lethal to bacteria, but all of them will induce the formation of thymine dimers. UV formation of a cyclobutane ring between the two adjacent thymines, called a thymine dimer. Without chemical and ionizing mutagens, the rate of mutagenesis would be so low, mutant yield would be low in the laboratory, and isolating them would be impractical.
Spontaneous mutations can occur when bases react with water or other natural species in the cellular environment. For example, cytosine deaminates to uracil, methylcytosine deaminates to thymine, adenine . Bases can tautomerize, switching from keto to enol form. Each form has a different base pairing property. For example, a guanosine in its keto form base pairs with cytosine, but a guanosine in its enol form will base pair with thymine. Thus, a switch from the keto to the enol form results in a G:C to A:T transition in one of the daughter cells. Mutagenic base analogs like 5-bromouracil tautomerize and cause transitions this way. However, DNA damage can involve more than just incorrectly incorporated bases. When double strand breaks or gaps occur, they are repaired either by non-homologous end joining (before replication) or homologous recombination (after replication, when sister chromatids can provide a template). In NHEJ, an exonuclease process the single stranded ends of the broken DNA, and ligases then directly join them together. The digestion of ends may result in the loss of nucleotides and mutations. Homologous recombination is less mutagenic because sister chromatids or other homologous regions are used as a template for repairing the gap.
Given that these changes to the DNA do not result in mutations unless they are fixed after a round of replication, the cell must repair the DNA before replication ends. This Monday night, we'll go over how the cell detects aberrations and repairs them!
Saturday, September 10, 2011
Hubble Telescope Discovered Dark Matter
Dark matter makes up about 73% of our universe, according to space.com.
Well, a discovery the Hubble Telescope was responsible for is dark matter, which has a unique composition compared to gas and dust in galaxies, and makes up most of the universe. A dark matter ring was discovered in 2007 at the site of two galaxy clusters’ collision, 5 billion light-years away from Earth. It was detected by the deflection, or bending of light in background galaxies when looking at the two galaxies’ collisions. The dark matter is probably a result of the high gravity following the collision. Evidence for dark matter, therefore, is indirect, only detectable when galaxies in the foreground caused distortions in the light of galaxies in the background.
Currently, the Hubble Space Telescope’s ACS is being used to survey dwarf galaxies 250 million light years away, in the Perseus Cluster. It is still too far away to detect small stars that may be too faint, but larger, more luminous stars are still readily detected. The Hubble Space Telescope revealed that at least 12 of the stars in the Perseus Cluster require dark matter. The stars appear smoother, scientists believe, because of the presence of dark matter, relative to the stars in spiral galaxies such as the Milky Way. The dark matter would protect these dwarf galaxies, whose stars have a high mass to light ratio (Hubble made it possible to calculate their mass by measuring the amount of X-ray radiation emitted by the hot gases of the stars), from being destroyed by outside gravitational pull. The discovery of this phenomenon would have been impossible without the incredibly high detail provided by a space telescope such as Hubble. As previously mentioned, ground telescopes have too much background light interference and not enough resolution for study of individual stars in galaxies outside our own. [1] [2]
Well, a discovery the Hubble Telescope was responsible for is dark matter, which has a unique composition compared to gas and dust in galaxies, and makes up most of the universe. A dark matter ring was discovered in 2007 at the site of two galaxy clusters’ collision, 5 billion light-years away from Earth. It was detected by the deflection, or bending of light in background galaxies when looking at the two galaxies’ collisions. The dark matter is probably a result of the high gravity following the collision. Evidence for dark matter, therefore, is indirect, only detectable when galaxies in the foreground caused distortions in the light of galaxies in the background.
Currently, the Hubble Space Telescope’s ACS is being used to survey dwarf galaxies 250 million light years away, in the Perseus Cluster. It is still too far away to detect small stars that may be too faint, but larger, more luminous stars are still readily detected. The Hubble Space Telescope revealed that at least 12 of the stars in the Perseus Cluster require dark matter. The stars appear smoother, scientists believe, because of the presence of dark matter, relative to the stars in spiral galaxies such as the Milky Way. The dark matter would protect these dwarf galaxies, whose stars have a high mass to light ratio (Hubble made it possible to calculate their mass by measuring the amount of X-ray radiation emitted by the hot gases of the stars), from being destroyed by outside gravitational pull. The discovery of this phenomenon would have been impossible without the incredibly high detail provided by a space telescope such as Hubble. As previously mentioned, ground telescopes have too much background light interference and not enough resolution for study of individual stars in galaxies outside our own. [1] [2]
1. Hubble Finds Ring of Dark Matter. 5 Oct 2010. Available:
2. Hubble Provides New Evidence for Dark Matter in Small Galaxies. 14 Nov 2010. Available:
Edit: If there's anything I want you to get from these recent posts on the Hubble, it's that funding for space programs helps us understand so many new things about the universe we live in and its beautiful physical laws. Dark matter would have probably remained a mystery if it had not been for the images from Hubble. Please write to your representatives urging them to continue supporting NASA or PBS, which has fascinating programs to educate us all.
Wednesday, September 7, 2011
Cepheids
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| The smaller of the two stars is a Cepheid variable. Credit: ESO/L. Calçada |
Charged couple devices on the Hubble are what helped us figure out the rate of expansion in the universe by using the opacity of stars near us. Dense stars have no temperature gradient, while less dense stars do. NICMOS and the infrared sensors were used to detect any temperature gradients in stars to determine their relative density. It detected stars that were variable between a dense and a less dense state, with observable changes in pressure and temperature. These stars are called Cepheids. This variation’s period was shown to be correlated with the apparent luminosity of the star, which increases and decreases at a regular interval.
The star’s relative distance can be determined now by detecting the change in the pulsating luminosity over time. Stars very far away are dimmer, and stars closer to us appear brighter. The scientists measured the differences between the brightness of different galaxies, cross-checked this data with other distance indicators, and determined how fast these galaxies are moving apart from each other. Cross-checking is necessary because other factors can affect the luminosity of stars, and having only one variable to determine the distance between stars would lead to many inaccuracies. For example, big stars are very dusty, but also very short-lived, which was a limitation for scientists, and gravity of different galaxies can affect the rate of movement, distorting the rate of expansion of the universe. It is possible for us to figure out the rate of expansion because the space telescope can adjust for the relative motion caused by the pull of gravity of galaxies nearby. It was a big step in the attempt to determine the rate of the expansion of the universe
Source:
WMP, the Expansion of the Universe. 12 Nov 2010. Available: http://map.gsfc.nasa.gov/universe/uni_expansion.html
Tuesday, September 6, 2011
TTAG In-Depth Look: Instrumentation on the Hubble Space Telescope
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| http://arstechnica.com/science/news/2010/04/hubble-turns-20-a-retrospective-in-pictures.ars (click to enlarge) |
The Hubble Space Telescope is responsible for many discoveries that revolutionized our view of the universe. A few observations here and there of phenomena in a minute portion of the sky is all that is needed to infer how our entire universe works. To illustrate, a few of Hubble's images lead to the discovery of dark matter. Yes, physics is that elegant.
Since its launch in 1990, the Hubble Space Telescope has provided the whole world with a detailed view of the universe that was never before possible. Given that the Hubble Space Telescope is outside of the Earth’s atmosphere, the images are clearer and sharper than any telescope located on the Earth’s surface. This is because the light that the ground telescope receives goes through air first. The molecules in the air distort and diffract light, and they absorb certain wavelengths of light, such as ultraviolet and gamma radiation. Space telescopes don’t have this problem outside of the atmosphere. Since it is in orbit above Earth’s atmosphere, there is no background light from Earth, and light is unaltered and undistorted by the molecular gases in our atmosphere.
The Hubble Space Telescope was launched into low Earth orbit by the space shuttle Discovery on April 25th 1990, It travels about 5 miles per second, takes 97 minutes to complete an orbit around Earth. [15] Even though it was meant to have been launched in 1983, it was delayed seven years because of financial constrains, and a cautious Congress and populace after the Challenger disaster a few years prior in 1989.
It is expected to re-enter into Earth’s orbit within the next few decades, due to orbital decay and drag, at which time a new space telescope is planned to replace it. Retrieval is considered not practical as it would risk the lives of people and would be too costly to do, as the shuttle program is already retired.
Instrumentation
The Hubble Space Telescope works to collect light from a wide spectrum and focus it, allowing us to see farther and clearer than the human eye possibly could. It is a Ritchey-Cretien Cassegrain reflector, meaning incoming light that travels from distant stars will come and bounce off a primary concave mirror and hit a secondary convex mirror, which focuses light through a small hole in the center of the primary mirror. The primary mirror’s diameter and size is one of the main determinants for how much light the telescope can collect, and compared to the large ground telescopes in observatory towers, the one on the Hubble would be considered small, at just 2.4 meters. [15] After incoming light passes through the hole in the primary mirror, it can be collected by the instrumentation on the Hubble Space Telescope. The mirrors must be very smooth, uniform don to 1/800,000th of an inch, and are treated with various coats to increase transmissibility of light, and to protect the mirror itself from warping due to solarization and aging due to ultraviolet light. The complex structure of the two mirrors, apertures, and trusses that support it is called the Optical Telescope Assembly, or OTA. [16]
Even small aberrations of the mirrors can cripple the Hubble Space Telescope. For example, after its launch, they discovered that the main mirror’s curvature had problems, but that was fixed after the first service mission in 1993. Since its launch, the Hubble Space Telescope’s fine instrumentation have required five different service missions: Service Mission 1 in 1993, Service Mission 2 in 1997, Service Mission 3A in 1999 to put in a new computer and gyroscopes, 3B in 2002 to replace the FOC with the Advanced Camera for Surveys, repair the NICMOS Cryocooler, and the most recent one, 4, in May 2009 to repair the Advanced Camera for Surveys, the Space Telescope Imaging Spectrograph, replace the Fine Guidance Sensoring System and two gyroscopes. [2][5][6][7][8] To maintain accuracy and for the data retrieved by the telescope be useful for scientific observation, constant upkeep and calibrations of the instruments must be made. The instruments themselves will now be described.
NICMOS and Cryocooler
NICMOS, or Near Infrared Camera and Multi-Object Spectrometer was installed on the Hubble Space Telescope in February of 1997, during the Servicing Mission 2. In short, it seeks out heat in the form of infrared radiation.
It is composed of three cameras, each with their own field of view, with 256x256 HgCdTe Rockwell sensor, sensitive to wavelengths of .8 micrometers to 2.5 micrometers. They are housed in a cryogenic dewar, maintaining a constant working temperature between 58 and 60 degrees Kelvin. The dewar is composed of three shields to keep NICMOS cool: the VCS, or Vapor Cooled Shield, the Thermo-Electric Cooled Inner Shield, and the Thermo-Electric Cooled Outer Shield. The hybrid nitrogen and aluminum dewar was supposed to keep it at just 58 degrees Kelvin, but a gap in the Vapor Cooled Shield resulted in an unanticipated heat load. Fluctuations in temperature where the cameras are housed means the cameras will need to calibrate more often.
This is where the Cryogenic Cooler comes into play, maintaining temperature stability within .5 degrees Kelvin. It circulates neon gas through a cooling loop, using high-speed centrifugal machines that do 7000 revolutions a second to compress gas, removing heat from the gas. [18]
NICMOS is useful because it captures information about infrared light, which reaches Earth from very far away, unaffected by interstellar dust, unlike visible light.
Advanced Camera for Surveying
The Hubble Telescope’s FOC was replaced by the ACS in 2002, during the Service Mission 3B. It has three cameras that are sensitive to a wide spectrum of light, from ultraviolet to near infrared (wavelengths of 1,200 to 10,000 angstroms), and since it has high contrast even near bright stars, it can be used to study galaxies and black holes very far away, where light from the ancient universe has just arrived at Earth. ACS has many components that make it versatile and useful for scientific observations. For instance, ACS is sensitive to ultraviolet light because it has a solar blind camera, or SBC, to block out visible light.
The Wide Field Channel of the ACS has two cameras with a resolution of 2048x4096 pixels each (for a total of 4k by 8k pixels), and its wide view frame is used to survey galaxies and the positions of stars within those galaxies. It is responsible for the Ultra Deep Field images of the universe.
The High Resolution Channel of the ACS has one camera with a resolution of 1024x1024, and is used primarily to detect ultraviolet light. The HRC has a coronagraph component that increases the contrast near stars tenfold. The High Resolution Channel allows us to “zoom” in, with a smaller field of view, but the images have a greater angular resolution. While the WFC and HRC are mainly on the red and blue regions of light, there’s a Multi-Anode Microchannel Array has no electronic noise and is sensitive to ultraviolet light, but not visible light. The images are 1k by 1k pixels, which is less than the WFC and HRC. Only the Solar Blind Camera is working right now. [10]
COS, STIS, and Ultraviolet Light
Spectrographs allows us to see precise information about incoming light that would otherwise be very faint and unusable. Incoming light is broken down to different wavelengths of light, and the amounts of each light at specific wavelengths are plotted on a graph. The light would have a unique fingerprint of different wavelengths, which can be studied.
COS, or the Cosmic Origins Spectrograph, looks at specific stars.
STIS, or Space Telescope Imaging Spectrograph, primarily has a larger field of view, focusing mainly on galaxies or a system of stars.
The Hubble Space Telescopes main mirrors are coated with magnesium fluoride 1 x 10-6 inches thick to improve the reflection of light at ultraviolet wavelengths. [16] Ultraviolet light, with a wavelength of 300 nanometers to 10 nanometers, is very high energy, so a special material that had a high energy gap and was resistant to absorbing the high energy from the photons was required. Magnesium fluoride not only helps maintain the integrity of ultraviolet light, the resistance to absorbing photon energy prevents warping of the mirrors due to solarization. [17][19]
Pointing and Guidance Systems
A system of fine guidance sensors, magnometers, solar sensors, and gyroscopes help keep the telescope position and point itself to collect light and data from specific areas in our universe. Since the Hubble Space Telescope is constantly moving in low orbit at a rate about 5 miles per second, a wealth of stabilizing components is needed to ensure the telescope is pointed at one spot for sufficient periods of time. The position and orientation of the telescope is also crucial because solar energy needs to hit the panels to power the telescope, and to keep the Sun’s heat from hitting only one side of the telescope. [19][4]
Currently, 3 gas-bearing gyroscopes rotate up to 19,000 rpm help stabilize the telescope, and with the help of up to three fine guidance sensors, the Hubble Space Telescope can help determine star locations that are 10 times more precise than a ground telescope. The guidance system also helps determine the position of stars, the distances between them, and the distance scale of the universe. Magnometers and CSS also help determine the telescope’s position relative to Earth’s magnetic field and the sun, respectively. Magnetic torquers and reaction wheel actuators help “lock” the telescope on to a planet or point in space. [4][19]
Design for Power
The Hubble Space Telescope is powered by 57 kilograms of nickel-hydrogen batteries when solar power is not available for 36 minutes in the Earth’s shadow. [3] Each orbit is 97 minutes, so it spends roughly 37 percent of its time without solar energy, so batteries are important. They must be able to power and sustain the telescope when solar power isn’t available. There are two modules consisting of a total of six batteries. Each module weighs 460 pounds and measures 36 inches long, 32 inches wide, and 11 inches high. There are three batteries per module, with each battery having 22 individual nickel-hydrogen cells placed in series.
Even though each battery has a total of 88 amp-hrs of capacity, on the Hubble Space Telescope, the practical maximum is 77 amp-hrs due to a limitation with heat dissipation. With a total of six batteries, the total energy supply becomes 450 amp-hrs. The batteries have lasted almost 13 years more than originally planned, 18 total. The ones that will replace them will be even better, built with the “wet slurry” process over the dry method. The “wet slurry” process allows for better porosity of the solid metallic powder over the dry method, in which the metallic powder is simply impacted into the battery cells. [3]
With the new batteries, the Telescope be used for normal scientific purposes on battery power for nearly 5 orbits, which is nearly 7.5 hours of operation, assuming the batteries are fully charged. [14] This means power for the Hubble Space Telescope is supplied redundantly in the sunlight, so if one source of power fails, the telescope will still be in working condition.
When the Hubble Space Telescope is within the day time portion of its orbit, about 61 minutes of the 97 minute orbit, it uses an array of four solar panels to power the telescope and to charge the batteries. They were replaced in the Servicing Mission 3B in 2002 with smaller, less flexible panels that produced less than a third more power than the pre-existing ones. Solar arrays supply 5k watts of energy to power the Hubble Space Telescope. This extra power supply makes running more instruments simultaneously on the telescope possible, so more institutions and scientists can use the telescope at the same time. [13]
1. Hubble Space Telescope Servicing. 5 Oct 2010. Available:
2. Servicing Misson 4. 5 Oct 2010. Available:
3. Servicing Mission 4 <Batteries>.
4. Servicing Misson 4 <Gyropscopes>. 5 Oct 2010. Available:
5. Servicing Mission 3B. 5 Oct 2010. Available:
6. Servicing Mission 3A. 5 Oct 2010. Available:
7. Servicing Mission 2. 5 Oct 2010. Available:
8. Servicing Mission 1. 5 Oct 2010. Available:
9. NASA Hubble. 5 Oct 2010. Available:
10. The Hubble Program – Technology. 5 Oct 2010. Available:
11. Hubble Finds Ring of Dark Matter. 5 Oct 2010. Available:
12. WMP, the Expansion of the Universe. 12 Nov 2010. Available: http://map.gsfc.nasa.gov/universe/uni_expansion.html
13. Hubble Servicing Missions. 25 Oct 2010. Available:
14. Hubble the Telescope. 25 Oct 2010. Available:
15. Hubble Telescope Essentials. 26 Oct 2010. Available:
16. Optical Assembly. 12 Nov 2010. Available:
17. Hubble Provides New Evidence for Dark Matter in Small Galaxies. 14 Nov 2010. Available:
18. NICMOS. 12 Nov 2010. Available:
19. Hubble – Fine Guidance Sensors. 5 Nov 2010. Available:
http://hubblesite.org/the_telescope/nuts_.and._bolts/instruments/fgs/index.php
Saturday, September 3, 2011
Question of the Day: Occupational Hazards
Question of the day for you folks: what kind of occupational hazards do you encounter at your job?
[Update: Deleted the post 9/10/11... just left the question!]
[Update: Deleted the post 9/10/11... just left the question!]
Thursday, September 1, 2011
Assay for β-galactosidase Specific Activity
As previously mentioned, β-galactosidase cleaves lactose into galactose and glucose. However, these products of the natural reaction are not easily quantifiable. That is why instead of lactose, we introduce ONPG to the cells. (ONPG, o-nitrophenyl-B-galactoside, is a substrate but not an inducer of the lac operon.) ONPG, colorless, is cleaved by β-galactosidase into galactose and orthonitrophenol, which is yellow, has a λmax of 420. The amount of o-nitrophenol can be quantitatively reported by looking at the change in the absorbance at 420 nm. The ΔA420/min is the total rate of the enzyme’s activity. To find the specific activity of β-galactosidase in relation to cell concentration, the rate is divided by the concentration of bacteria cells. Thus, the specific activity can be expressed by the following equation: ΔA420/min/OD600/ml. If the culture is not in log phase and in the death phase, dead cells contribute to the optical density but they can’t produce β-galactosidase to contribute to the total activity, leading to a deflated specific activity value for the enzyme.
Let's go it over step by step, shall we? I'll walk you through it.
A culture in the log phase is needed, as inducing gene expression works best during this phase. Inoculate liquid broth with E. coli and grow it at 37⁰ C. The broth shouldn’t have any glucose, as this would block any lacZ expression. Using the broth as a blank, transfer the culture into cuvettes and take the OD600 of the culture until it reads approximately .5-.6. In a tube with the culture, add the phosphate Z buffer, which keeps β-galactosidase working efficiently. Add a drop of sodium docecyl sulfate (SDS) and a drop of chloroform, which will lyse the cells, stop translation, but leave β-galactosidase intact and available for ONPG hydrolysis. Keep this in a room temperature water bath until the temperature has equilibrated. It should be stable for a while, preventing time errors. Add ONPG to each tube being assayed. This will be time 0, the start of the reaction. An Eppendorf tube with distilled water, Z buffer, and ONPG should be used as a control to make sure hydrolysis of ONPG is because of the enzyme, and not a spontaneous reaction in the mixture. If, after 5 minutes, the tubes turn yellow too quickly, the bacterial culture should be diluted and re-assayed, because that small window of time means small time errors will skew the results significantly. In addition, there needs to be an excess of ONPG during the assay, to ensure that the rate of hydrolysis is limited by, and therefore determined by, the enzyme concentration. If not enough ONPG is available, the increase in A420 would not correlate with increases in expression of galactosidase. The start of induction for this enzyme takes about 15-30 seconds, with a logarithmic increase when plotted against time, so sometimes reactions can go to completion is inappropriate amounts of ONPG are used. Use sodium carbonate (Na2CO3) once the yellow color is observed to stop the reaction. Na2CO3 inhibits β-galactosidase activity, thus stopping ONPG hydrolysis. Take the tubes and centrifuge them for 10-15 minutes at 5,000 g. Transfer the supernatant to cuvettes and measure the OD420. Visually confirm the readings are reasonable. A420 should be higher the darker the yellow. If the pellet and supernatant are not separated properly, the cells will scatter the light and invalidate the readings. During the entire reaction period, the temperature must be kept constant, because variable temperatures will affect enzyme activity and absorbance values.
The change in A420 can be converted to moles of ONGP converted to ONP+ by using Beer’s law and plugging in the molar extinction coefficient for ONP+, 4500/M/cm. The moles of ONP+ converted can be divided by the turnover rate of β-galactosidase to find out how much β-galactosidase is active.
The assay is used to determine relative expression of the lacZ gene and the specific activity of β-galactosidase, the lacZ gene product, in a bacterial culture. In uninduced cultures vs. fully induced cultures, there is a 1000% difference in β-galactosidase activity. Using this assay, the amount of time that it takes for the lac operon to become fully induced in a culture and be determined. Most importantly, we can manipulate the operon and insert a set of other structural genes downstream of the lac promoter by homologous recombination. Presence of a neoR cassette, too, allows us to select for these transgenic cells. Since the sequence will under the promoter’s regulation, the expression of the genes inserted is proportional to the expression of lacZ. Thus, we can indirectly determine expression of the inserted sequence by assaying B-galactosidase activity.
Let's go it over step by step, shall we? I'll walk you through it.
A culture in the log phase is needed, as inducing gene expression works best during this phase. Inoculate liquid broth with E. coli and grow it at 37⁰ C. The broth shouldn’t have any glucose, as this would block any lacZ expression. Using the broth as a blank, transfer the culture into cuvettes and take the OD600 of the culture until it reads approximately .5-.6. In a tube with the culture, add the phosphate Z buffer, which keeps β-galactosidase working efficiently. Add a drop of sodium docecyl sulfate (SDS) and a drop of chloroform, which will lyse the cells, stop translation, but leave β-galactosidase intact and available for ONPG hydrolysis. Keep this in a room temperature water bath until the temperature has equilibrated. It should be stable for a while, preventing time errors. Add ONPG to each tube being assayed. This will be time 0, the start of the reaction. An Eppendorf tube with distilled water, Z buffer, and ONPG should be used as a control to make sure hydrolysis of ONPG is because of the enzyme, and not a spontaneous reaction in the mixture. If, after 5 minutes, the tubes turn yellow too quickly, the bacterial culture should be diluted and re-assayed, because that small window of time means small time errors will skew the results significantly. In addition, there needs to be an excess of ONPG during the assay, to ensure that the rate of hydrolysis is limited by, and therefore determined by, the enzyme concentration. If not enough ONPG is available, the increase in A420 would not correlate with increases in expression of galactosidase. The start of induction for this enzyme takes about 15-30 seconds, with a logarithmic increase when plotted against time, so sometimes reactions can go to completion is inappropriate amounts of ONPG are used. Use sodium carbonate (Na2CO3) once the yellow color is observed to stop the reaction. Na2CO3 inhibits β-galactosidase activity, thus stopping ONPG hydrolysis. Take the tubes and centrifuge them for 10-15 minutes at 5,000 g. Transfer the supernatant to cuvettes and measure the OD420. Visually confirm the readings are reasonable. A420 should be higher the darker the yellow. If the pellet and supernatant are not separated properly, the cells will scatter the light and invalidate the readings. During the entire reaction period, the temperature must be kept constant, because variable temperatures will affect enzyme activity and absorbance values.
The change in A420 can be converted to moles of ONGP converted to ONP+ by using Beer’s law and plugging in the molar extinction coefficient for ONP+, 4500/M/cm. The moles of ONP+ converted can be divided by the turnover rate of β-galactosidase to find out how much β-galactosidase is active.
The assay is used to determine relative expression of the lacZ gene and the specific activity of β-galactosidase, the lacZ gene product, in a bacterial culture. In uninduced cultures vs. fully induced cultures, there is a 1000% difference in β-galactosidase activity. Using this assay, the amount of time that it takes for the lac operon to become fully induced in a culture and be determined. Most importantly, we can manipulate the operon and insert a set of other structural genes downstream of the lac promoter by homologous recombination. Presence of a neoR cassette, too, allows us to select for these transgenic cells. Since the sequence will under the promoter’s regulation, the expression of the genes inserted is proportional to the expression of lacZ. Thus, we can indirectly determine expression of the inserted sequence by assaying B-galactosidase activity.
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