Author Archives: natasha tripp

Assignment -> bees

Leave a reply
  1. What could be the underlying mechanism driving the different developmental trajectory of the organisms studied in today’s class?

Since worker and queen bees have practically identical genotypes, some sort of environmental cue must be affecting the phenotypes arrived at from the same genotype. These environmental factors most likely affect epigenetic factors like DNA methylation, histone methylation and histone variants to change the dosage of certain gene products produced that results in different physical features. (APPAREANTLY DIET IS THE KEY TO THE DIFFERENT DEVELOPMENTAL TRAJECTORIES).

  1. a) With one or two partners, take two minutes to come up with a definition of “epigenetics”:

Heritable modifications to gene expression that are also unrelated to the DNA sequence.

b) After listening to other classmates’ ideas, provide a more complete (if necessary) definition of “epigenetics”:

After hearing Pam’s description of the unicellular protist that can be physically manipulated such that their offspring inherit the physical manipulation, it might be the case the epigenetics isn’t specific to modifications to gene expression but also more gross anatomy manipulations.

  1. a) What are different mechanisms that can affect developmental trajectory, and that could be affected/directed by an “outside factor”?

Signaling pathways that affect that production of hormones that have downstream affects on gene regulation.

On/Off gene switches that result in methylation patterns amongst other manipulations to chromatin structure.

b) What are the mechanisms that can affect chromatin structure?

Supercoiling

Histone variants

Histone modifications

Locations in the nucleus

DNA modifications

Chromatin remodeling factors (slide nucleosome along the DNA)

  1. What would you predict about gene expression patterns in the two distinct developmental trajectories if epigenetics is driving the phenotype?

Each distinct developmental trajectory will have differential gene expression of particular genes related to development of size, life span and reproductive organs since queen bees are larger, live longer and can reproduce whereas the worker bees are smaller, live shorter lives and cannot reproduce.

  1. Researchers (Grozinger et al., 2007) actually checked… what do you notice about the gene expression patterns in individuals following each of the two developmental trajectories?

Genes involved in reproduction and longevity are upregulated in Queen bees and genes involved in foraging behavior are upregulated in worker bees. Furthermore, genes involved in foraging behavior are down regulated in Queen bees and genes involved in reproduction and longevity are down regulated in worker bees.

  1. What kind of protein/factor could be a key component of the epigenetic control of developmental trajectories? How would you test your hypothesis?

Since honey bees have a fully functional methylation system, the differential gene expression might be the result of differential methylation patterns. Therefore, the key component of epigenetic control might be a DNA methyltransferase.

  1. What did Kucharski and colleagues find, and what does their experiment suggest?

Kucharski et al found that knocking down the DNA methyltransferase Dnmt3 by injection of siRNA results in an increased number of ovarioles per ovary that is a distinguishing feature of the queen phenotype. Knockdown of Dnmt3 mimics royal jelly in producing the queen phenotype. Therefore, there might be a compound in royal jelly that has similar effects as knocking down Dnmt3 has.

  1. a) What component of the food in question is most likely to affect gene regulation?

Protein since enzymes are proteins and enzymes are the most likely compound to have activity that can regulate DNA methylation and other epigenetic markers.

b) How does the food in question activate a transcriptionally silenced gene?

Royal jelly acid or 10HDA activates transcriptionally silenced genes by removing methyl groups from the DNA of a gene and, also, by inhibiting the deactylation of histones of the nucleosomes of the genes of interest.

 

Techniques -> qPCR

Leave a reply

TECHNIQUES SPEED-DATING: Quantitative Polymerase Chain Reaction

Names and contributions of group members:

Kenrick Ocampo-Tan, Natasha Tripp, Lorenzo Luis Casal, Melissa Chen, Kimmy Wong

Technique chosen: Quantitative PCR and its derivatives

What does this technique ‘do’?

  • Quantitative polymerase chain reaction estimates the starting copy number of a DNA/mRNA segment
    • Can be estimated using the final product concentration after ‘X’ cycles (end-point qPCR) or by detecting the amount of products generated after each cycle (real-time qPCR)1

What applications are this technique employed for?

  • Estimating DNA copy number or detecting DNA from a sample2
  • Gene expression analysis by quantifying mRNA (RT-qPCR)2,4

What questions (give a couple of examples) relating to gene regulation and/or development can be addressed using this technique?

  • How does the copy number of a certain gene compare with others? (qPCR)
  • What housekeeping genes are active during specific time in development of fetal animals? (RT-qPCR)
  • What genes are used in development but rarely expressed in adulthood? (RT-qPCR)
  • Is there differential expression of genes under different stress conditions? (RT-qPCR)

What critical reagents are required to use this technique?

  • Both qPCR and RT-qPCR require standard PCR reagents (Forward and reverse primers, dNTPs, thermophilic polymerase, Mg2+, appropriate buffers)
    • qPCR also requires DNA template and either a dye or probe that fluoresces when hydrolyzed or hybridized4
    • RT-qPCR requires reverse-transcriptase (and other standard RT-PCR reagents)  in addition to all of the above
  • real-time PCR requires a PCR machine capable of detecting the amount of product after each cycle

What critical information is required to be able to employ this technique?

  • Sequence data to be able to create primers
  • Proper annealing temperature for reaction specificity

References:

  1. Heid, Christian A., Junco Stevens, Kenneth J. Livak, and P. Mickey Williams. “Real time quantitative PCR.” Genome research 6.10 (1996): 986-994. Web. 01 Feb 2015.
  2. Karlen, Yann, Alan McNair, Sébastien Perseguers, Christian Mazza, and Nicolas Mermod. “Statistical significance of quantitative PCR.” BMC bioinformatics 8.1 (2007): 131. Web. 01 Feb 2015.
  3. Lederman, Lynne. “QPCR.” BioTechniques 47.4 (2009): 817-19. Web. 01 Feb 2015.
  4. VanGuilder, Heather, Kent Vrana, and Willard Freeman. “Twenty-five Years Of Quantitative PCR For Gene Expression Analysis.” Biotechniques 44.5 (2008): 619-26. Web. 01 Feb 2015.

 

 

Wk 13 Learning Journal #5

Leave a reply

A. Three main things in BIOL463

Please describe, briefly, the three things that you learned in BIOL463 and that you consider to be the “most important” ones.

1. I learned to much more clearly make the very important distinction between necessary and sufficient when looking at data.

2. I learned the difference between what the data shows, conclusions, and models.

3. You can’t always trust the figure legends or conclusions that papers make

 B. Identify types of knowledge

For each of your three “things”, please try to identify what type of knowledge it represents (Factual, Conceptual, Procedural/Skills, Metacognitive).

1 and 2 are somewhat procedural and conceptual. 3 is metacognitive.

C. What makes “things” stand out for you

For each of your three “things”, please indicate what made it stand out for you.

  • This was introduced to me in Biol 200 but it seemed a lot simpler then. In this class looking at data from papers and making necessary/sufficient statements seemed more difficult in the beginning because it was using data that I had not seen before.
  • Again, in Biol 200 we had to make a distinction between what the data show and what they mean, but in this class I then had to make the further distinction to a model, where conclusions are combined to explain the data. This step is much more difficult in my mind and I thought we got a lot of practice and feedback in this course.
  • There were a couple of instances in class where I was tripped up a bit in my thinking because I took the figure legend at face value and didn’t think through what was actually being presented.

D. Relevance/use

Please describe, briefly, what you expect each of these three “things” to be useful or relevant for.

These three things will be useful in a lot of ways, including thinking about any further research I do, reading papers for interests sake, or solving problems in everyday life.

E. Three main things in your undergraduate education

Please describe, briefly, the three things that you learned during your undergraduate education and that you consider to be the “most important” ones. Why do you find them so important?

  1. Be careful to look at where information comes from. Is it from a credible source? From a peer review journal?: This is incredibly important when looking at information on the internet, often statements are made that may not be entirely what was found using credible peer reviewed research or there might not be any research on something that is being stated as fact. It might be a little pessimistic but in today’s society its usually good to be a bit skeptical of information that someone is talking about, writing about, or discussing if they cannot show where they got that information from
  2. A lot is known but there are a huge number of unanswered questions that still need to still be addressed and explored: I found that this was, to me at least, the central theme of 2nd I was shocked by the number of times there was a statement like “we know this is true for this species, but we don’t know for others” in my microbiology textbook. I’ve come to realize that the more I know, the more questions I can come up with because of connections to other topics, classes, and sometimes random things.
  3. Teamwork is so important, and may be one of the most important things in science: Collaborative science is able to produce so much more information and ideas than people by themselves and the body of work that makes up what we know about science today was developed by an incredibly large number of people. This is also true in smaller ways in that labs that are able to assist each other and work in a creative and encouraging environment may make more progress than those that are at odds with each other.

Final projects -> final

Leave a reply

Question

Do BPA analogs have similar adverse effects as BPA on the structure and function of the developing mammalian ovary in vivo?

Introduction

Endocrine disrupting chemicals (EDCs) are compounds that can mimic or block the action of endogenous hormones in the body. These natural or synthetic chemicals can have major effects on development, fertility, and may be associated with cancers of the reproductive tract and mammary glands. Developmental exposures have been shown to induce meiotic defects and cause incorrect follicle formation in the developing ovary (Hunt & Hassold, 2008). These defects have long term implications and may adversely affect fertility and reproductive lifespan (Hunt et al., 2012). Developmental exposures seem to have a larger effect than exposures in adulthood, which can cause dose dependent and sex dependent changes to body weight (Rogers et al., 2013; Rubin & Soto, 2009). Bisphenol A (BPA) is an estrogen mimicking EDC that is commonly found in cans, plastic food and drink containers, dental materials, and receipts, leading to a relatively constant human exposure (Maffini et al., 2006). Recently, increasing numbers of animal studies have found that even at low levels BPA has detrimental effects on meiotic events and follicle formation in the developing ovary in both mouse and primate models (Hunt & Hassold, 2008; Hunt et al., 2012). All research looking into BPA’s effect on the developing ovary has been done using animal models because of ethical reasons that prevent the study of humans in this way.

BPA has been found to bind to estrogen receptors (ERα and ERβ) leading to dysfunction in the normal regulation of genes affected by these receptors. These receptors act as transcription factors that travel into the nucleus and cause changes to gene expression. ERα is expressed in theca cells (support cells for each follicle) and in the ovarian stroma during ovary development and in adulthood (Sar & Welsch, 1999). ERβ is expressed in granulosa cells of developing follicles (Sharma et al., 1999). Mouse knockouts of ERα (ERKO) and ERβ (ERBKO) have very different phenotypes. ERKOs have a phenotype where follicles appear to develop normally but then never ovulate or produce the correct outer covering Also, individuals have large hemorrhagic cysts, and stromal hyperplasia, as well as an excess of other circulating hormones like luteinizing hormone, testosterone, and estrogen (Abbot et al., 2006). This shows that ERα is necessary for regulation of the effect of other hormones in the ovary (Couse & Korach, 2001). ERBKO exhibits a phenotype where individuals are able to ovulate and produce pups but have fewer ovulation events and smaller litter sizes (Abbot et al., 2006). This shows that ERβ is necessary for mediation of estrogen’s effect on the ovary in ways related to maturation of follicles (Emmen et al., 2005). BPA’s effect on these receptors leads to changes in gene expression and regulation in the ovary (Zhou et al., 2008). BPA has an agonist effect on ERβ and has both an agonist and an antagonist effect on ERα (Kuiper et al., 1998; Hiroi et al., 1999). As the phenotypes of the ER knockouts are known, the overall phenotype of BPA exposed individuals can be split into changes caused by activation of ERβ and variable inactivation and activation of ERα. Activation of ERβ likely causes the misregulation of follicle maturation and formation, and BPA’s variable action on ERα likely causes differences in amount of stromal proliferation, and the potential presence of cysts and abnormally small oocytes.

Due to an increase in our understanding of the detrimental effects of BPA, analogs of BPA are beginning to be used in plastics manufacturing instead. Less is known about these derivatives of BPA, though recent studies have determined that many of these compounds have similar effects on estrogen receptors (Stossi et al., 2014). Two of the many BPA analogs that are commonly used are Tetrachlorobisphenol A (TCBPA) and Bisphenol S (BPS). TCBPA is an ERα agonist and BPS has agonistic effects on both receptors, with a higher activity in binding to ERβ (Table 1) (Li et al., 2010; Molina-Molina et al., 2013). As these compounds also have estrogenic effects, they will likely produce a phenotype in the developing ovary. If TCBPA and BPS have similar negative effects as BPA, then these analogs should not be used in manufacturing and other non-BPA-like alternatives should be developed. Currently the analogs are being used instead of BPA and manufacturers are calling the resulting plastics “BPA-free” (Yang et al., 2011).

 

EDC Effect on ERα Effect on ERβ
BPA +/- +
TCBPA + None
BPS – –

Table 1. Effect of BPA and two analogs on estrogen receptors ERα and ERβ. (+ signifies agonist effect and – signifies antagonist effect)

 

Hypotheses and Predictions

I hypothesize that both TCBPA and BPS will cause abnormal phenotypes in developing ovaries due to their action on estrogen receptors. I predict that TCBPA will likely have little effect on the formation of follicles but will cause decreases in the amount of stromal cells and an increase in the number of small, potentially non-functional, oocytes. Also, BPS will likely cause changes in follicle maturation and decrease stromal proliferation and increase the number of small oocytes.

Experimental approach

In order to investigate this question I will use a mouse model system. Benefits of a mouse model include ease of housing, lower expenses, quicker reproduction, and the ability to produce more than one offspring per pregnancy; however, there are some differences in pregnancy progression that make this model slightly less applicable to humans when compared to results from primate model studies. Hunt et al. (2012) found that primates exhibited a very similar phenotype in the developing ovary as mice when exposed to BPA, so there is merit to performing initial studies using a mouse model. As there are no other in vivo studies that look at the effects of TCBPA and BPS, using a mouse model will give a solid foundation for future primate studies that will be more applicable.

I will have four treatment groups: control (no treatment), BPA, TCBPA, and BPS. Each treatment group will consist of at least three female mice that will have a device that releases a continuous low level dose of either BPA, TCBPA, or BPS implanted subcutaneously. The implant consists of rubber tubing that contains the chemical (Hunt et al., 2012). A pilot study will be conducted to make sure that dosage levels are close to the normal serum concentrations found in humans (Hunt et al., 2012). Control females will have the device implanted but without the chemical additives. The implants will be inserted pre-pregnancy and dosage will continue throughout pregnancy. This method is the most similar to normal human exposure and may give the most realistic results (Hunt et al., 2012). The mice will then be mated and embryos will be harvested at mid and late gestation, the beginning of meiotic division and follicle formation respectively.

Female embryos will be studied to determine the effect of BPA analogs on chromosome behavior during meiosis and follicle formation in the developing ovary. The developing ovaries will be removed and sectioned in preparation for analysis at each gestational stage. Chromosome behaviour at early meiosis will be observed by performing immunofluorescence imaging of ovaries at mid gestation. SYCP3, CREST antiserum, and MLH1 will be tagged using fluorescent antibodies. SYCP3 is a symaptonemal complex protein, CREST interacts with centromeres, and MLH1 is a component of recombination events (Hunt et al., 2012). Synaptic defects can be observed by looking for increased number of recombination events, using points of concentrated MLH1 as a proxy. Chromosome associations can be observed by looking for clusters of CREST that mark centromeres interacting. Mice chromosomes are all telocentric, the centromere is near the telomere, so observation of end-to-end chromosome interactions are easy to see in this way. Tagging these proteins means that interactions can simply be counted and then compared. Follicle formation at late gestation will be investigated by using light microscopy with hematoxylin and eosin staining on serially sectioned ovaries and then observing or counting any follicular abnormalities that result. For instance, Hunt et al. (2012) counted multi-oocyte follicles and nests of unenclosed oocytes. Normally follicles only contain one oocyte and only a few, if any, oocytes are not enclosed within follicles. Control ovary immunofluorescence images and sections will be used to compare normal occurrence of abnormalities to the number seen in individuals treated with BPA, TCBPA, and BPS. Statistical analysis can then be performed to determine if the abnormalities are significantly higher in the treated groups than in the control group. These methods allow observation of chromosome behavior and ovarian structures after in vivo exposure to EDCs.

Possible outcomes

There are many possible outcomes for this experiment:

  1. TCBPA and BPS may not show gross phenotypic changes in the developing ovary. Further study may then be needed to identify if gene expression and hormone regulation are affected without causing large outward changes. This result may also be due to the dose given to the pregnant mice. If this result occurs, then different doses should be used to see if there is a point at which TCBPA and BPS do have an effect on the structure of a developing ovary.
  2. TCBPA and BPS may show novel phenotypes (when compared to BPA) in the developing ovary due to their different effects on the ERs. Depending on the degree of effect, there may be variation in the phenotype produced. These phenotypes will likely be in chromosome behaviour and follicle formation. As stated in my prediction, I think that TCBPA will cause changes in the number of stromal cells, leading to a decrease in ovary size, and an increase in the number of abnormally small oocytes that may affect future reproduction. BPS will likely cause changes in follicle formation and maturation, as well as decrease stromal proliferation and increase the number of small oocytes.
  3. TCBPA and BPS may show phenotypes in the developing ovary that are similar to those of BPA. This may be due to other unknown effects that the new chemicals may have on regulation of ovary growth and maturation. The severity of the phenotype from the analogs may differ from those produced by BPA because of different levels of activity when binding to the ERs or to other receptors.

Lay person summary

Hormones are very important molecules in the normal growth and function of male and female reproductive tracts. Natural and synthetic chemicals can mimic the function of hormones in the body by interacting with normal hormone pathways; these chemicals are called endocrine disrupting chemicals (EDCs). BPA is a chemical that is used to make plastics and other materials found in food and drink containers, the inside of cans, and receipts. In recent years, this chemical has been found to be an EDC. BPA mimics the function of estrogen and as such has detrimental effects on the developing ovary, where the correct levels of estrogen is important for proper growth. More recently, chemicals derived from BPA (BPA analogs) have started to be used by manufacturing companies instead of BPA, due to the increasing evidence for health concern; however, little is known about the effects of the analogs on development. I will perform an experiment on mice because of ethical constraints that prevent the study of human developing embryos. I will expose female mice to continuous low levels of BPA and two analogs, TCBPA (Tetrachlorobisphenol A) and BPS (Bisphenol S), throughout pregnancy. I will then observe the structure of the embryos’ developing ovaries using techniques that allow observation of how the chromosomes interact and how the ovaries’ cells develop. This topic is interesting because these compounds could have detrimental effects on human reproduction and fertility in the future, as the next generation of humans is being born to mothers that have been exposed to BPA, TCBPA, BPS, and other analogs.

Literature cited

Abbott, D. H., Padmanabhan, V., & Dumesic, D. A. (2006). Contributions of androgen and estrogen to fetal programming of ovarian dysfunction. Reprod Biol Endocrinol, 4(17), i0006-3363.

Couse, J. F., & Korach, K. S. (2001). Contrasting phenotypes in reproductive tissues of female estrogen receptor null mice. Annals of the New York Academy of Sciences, 948(1), 1-8.

Emmen, J. M., Couse, J. F., Elmore, S. A., Yates, M. M., Kissling, G. E., & Korach, K. S. (2005). In vitro growth and ovulation of follicles from ovaries of estrogen receptor (ER) α and ERβ null mice indicate a role for ERβ in follicular maturation. Endocrinology, 146(6), 2817-2826.

Hiroi, H., Tsutsumi, O., Momoeda, M., Takai, Y., Osuga, Y., & Taketani, Y. (1999). Differential interactions of Bisphenol A and 17β-estradiol with estrogen receptor α (ERα) and ERβ. Endocrine journal, 46(6), 773-778.

Hunt, P. A., & Hassold, T. J. (2008). Human female meiosis: what makes a good egg go bad?. Trends in Genetics, 24(2), 86-93.

Hunt, P. A., Lawson, C., Gieske, M., Murdoch, B., Smith, H., Marre, A., … & VandeVoort, C. A. (2012). Bisphenol A alters early oogenesis and follicle formation in the fetal ovary of the rhesus monkey. Proceedings of the National Academy of Sciences, 109(43), 17525-17530.

Kuiper, G. G., Lemmen, J. G., Carlsson, B. O., Corton, J. C., Safe, S. H., van der Saag, P. T., … & Gustafsson, J. A. (1998). Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor β. Endocrinology, 139(10), 4252-4263.

Li, J., Ma, M., & Wang, Z. (2010). In vitro profiling of endocrine disrupting effects of phenols. Toxicology in vitro, 24(1), 201-207.

Maffini, M. V., Rubin, B. S., Sonnenschein, C., & Soto, A. M. (2006). Endocrine disruptors and reproductive health: the case of bisphenol-A. Molecular and cellular endocrinology, 254, 179-186.

Molina-Molina, J. M., Amaya, E., Grimaldi, M., Sáenz, J. M., Real, M., Fernández, M. F., … & Olea, N. (2013). In vitro study on the agonistic and antagonistic activities of bisphenol-S and other bisphenol-A congeners and derivatives via nuclear receptors. Toxicology and applied pharmacology, 272(1), 127-136.

Rogers, J. A., Metz, L., & Yong, V. W. (2013). Review: Endocrine disrupting chemicals and immune responses: a focus on bisphenol-A and its potential mechanisms. Molecular immunology, 53(4), 421-430.

Rubin, B. S., & Soto, A. M. (2009). Bisphenol A: Perinatal exposure and body weight. Molecular and cellular endocrinology, 304(1), 55-62.

Sar, M., & Welsch, F. (1999). Differential expression of estrogen receptor-β and estrogen receptor-α in the rat ovary. Endocrinology, 140(2), 963-971.

Sharma, S. C., Clemens, J. W., Pisarska, M. D., & Richards, J. S. (1999). Expression and Function of Estrogen Receptor Subtypes in Granulosa Cells: Regulation by Estradiol and Forskolin 1. Endocrinology, 140(9), 4320-4334.

Stossi, F., Bolt, M. J., Ashcroft, F. J., Lamerdin, J. E., Melnick, J. S., Powell, R. T., … & Mancini, M. A. (2014). Defining estrogenic mechanisms of bisphenol A analogs through high throughput microscopy-based contextual assays. Chemistry & biology, 21(6), 743-753.

Yang, C. Z., Yaniger, S. I., Jordan, V. C., Klein, D. J., & Bittner, G. D. (2011). Most plastic products release estrogenic chemicals: a potential health problem that can be solved. Environmental Health Perspectives, 119(7), 989.

Zhou, W., Liu, J., Liao, L., Han, S., & Liu, J. (2008). Effect of bisphenol A on steroid hormone production in rat ovarian theca-interstitial and granulosa cells. Molecular and cellular endocrinology, 283(1), 12-18.

Assignment -> BWS and SRS

Leave a reply
  1. Compare and contrast the phenotypes of the SRS vs. BWS patients (you will need to look at Table 1). Do you notice any trends? Knowing that all the patients studied in this article have mutations in the same imprinted cluster, what could explain the differences in phenotypes?

The SRS patient had low birth weight, low birth length, had post and pre natal growth failure, facial defects, digit deformities, and had nervous muscle deficiencies. The BWS patients were generally large at birth, maintain large stature, and do not have facial or digit deformities. The SWS patient has a duplication of all of the genes between ICR1 and 2, but the methylation pattern is the same meaning there is no inhibition by the lnRNA in ICR2. This means that there would be an increased amount of CDKN1C which is a cell growth inhibition, leading to growth deficiencies. The BWS patients have a mircoduplication that results in a truncated lnRNA that inhibits expression of the growth inhibitor, leading to an increase in growth and large size.

  1. The authors report using OMIM to obtain some information for their research. Take a few minutes to look up Kcnq1ot1 on OMIM and see what information you get.

Maps to chrom 11p.15.5, lnRNA, overlaps KCN1 in antisense

3. Look at the pedigree in Figure 1.

a) What can immediately be concluded about BWS (even without knowing who inherits the mutant allele from whom)?

The syndrome is probably not recessive autosomal and probably not X linked. Individuals with the syndrome are fertile, and the syndrome is not extremely lethal.

b) II-2 and II-4 both have BWS, and both have one child with BWS and one child without BWS. Briefly explain how this is possible.

If the affected mother passes on the allele that she inherited from her father (with the correct maternal methylation) then the allele will be normal resulting in a normal phenotype.

  1. Briefly describe the mutation detected in the BWS patients and the mutation detected in the SRS patient, and their respective effects at the molecular, cellular, and organismal levels (use figures 2, 3, and 8, as well as your answer to Question 1).

NOTE: molecular level = DNA sequence, DNA methylation, gene expression; cellular = proteins present in the cell, potential effects on the cell; organismal = effects on the entire organism.

SRS – 1.2 Mb duplication and inversion of the entire 11p15.5 region, methylation is consistent with maternal methylation (ICR1 is not methylated and ICR2 is methylated), the lnRNA is not expressed on the maternal chromosome, there is a double dosage effect of the genes in the 11p15.5 region on the maternal copy, increased cell growth inhibitor decreases cell growth, has the syndrome phenotype (low birth weight, low birth length, had post and pre natal growth failure, facial defects, digit deformities, and had nervous muscle deficiencies)

BWS – 160 kb duplication and inversion of the ICR2 and part of the KCNQ10TI sequence, methylation on the original copy is normally methylated but the partial RNA sequence is un-methylated, there is expression of the partial lnRNA leading to the down regulation of the cell growth inhibitor, cells grow larger, the individual is larger

  1. Explain what the data in Figure 7 show, and how you interpret them.

BWS patients have increased expression of the lnRNA and this is due to increased expression on the maternal allele. Paternal lnRNA expression is unchanged among individuals. The control individual only shows expression of one allele, while the DNA sequence shows that there are 2 different alleles present. The BWS patients show Chirp sequences from both alleles, meaning that there is both maternal and paternal expression of the lnRNA.

  1. What valuable fundamental information was gained about imprinting control regions through the study of these patients?

Having a sequence in cis to the ICRs does not necessarily mean that it will be properly imprinted. This is shown by the improper imprinting of the duplicated region in the BWS patients.

 

Assignment -> lacZ transgene

Leave a reply
  1. Recall the general rule, “Figure 1 is often the most important figure in the paper”. Referring to Figure 1:

1a)  What transgenic lines did the author use (do these lines look somewhat familiar)? Hoxd9/lacz (HoxDrel5) and HoxDInv(rel5-Itga6). These lines look similar to the line we discussed on Friday where there was also a large inversion.

b) What do the data show (Panel C)? There is normal expression of both parental copies without the inversion. Inversion causes loss of expression when inherited from the mother.

c) What is striking/unexpected about the results, and why? F1 progeny either had strong or almost no expression of lacZ. This indicates that the inversion places the lacZ sequence into a region where it is then maternally imprinted. This is striking because this is very site-specific.

d) What conclusions do you make from the data? Maternal imprinting is position dependent and occurred near the Itga6 locus.

2. How did the authors show that the observed imprinting is lacZ-specific and position/site-specific? Do you agree with their data interpretation and with their conclusion? The researchers used a different mouse line that has HoxD11 replacing the HoxD/lacZ transgene in the inverted sequence. Then they looked at the expression of HoxD11. There was no change when the inverted allele was maternally or paternally passed down, so when lacZ is not present there is no imprinting of the allele. The position specific aspect is shown in Figure 1. Their data interpretation and conclusion sound plausible.

3. Refer to Figure 2.

a) Briefly explain how to “read” the diagrams shown (i.e. what do the rows of circles represent, what do the white vs. black circles represent). Circles represent potential methylation sites at C residues. White circles are un-methylated and black circles are methylated.

b) What do the data in Figure 2 show? Methylation in escapers is much more variable. Maternal inverted alleles are heavily methylated and paternal inverted alleles are mostly un-methylated. This pattern is also seen in eggs and sperm. D shows the normal amount of methylation in the non-inverted line.

c) Why aren’t there a “paternal/+” and a “maternal/+” groups for sperm and oocytes? Sperm and eggs are haploid, so only have one allele. This only shows the methylation that is sex specific.

d) What are “escaper” embryos, and how were they identified prior to bisulphite sequencing? These embryos had some lacZ expression when the inverted reporter as maternally transmitted. Other maternal/- embryos had little to no expression of lacZ.

  1. What was the purpose of the authors’ ChIP experiments, and why did they choose to look for specific histone modifications? What did they find? (Expected answer: max two sentences)

The purpose of ChIP was to look at which regions were associated with H3K27me3. They found that the histones associated with the HoxD9 promoter and lacZ were heavily methylated compared to wild type.

  1. What does Figure 4 show? (Please describe/summarize its content including specific information).

A shows a strong interaction between Dlx1/Dlx2 and the transgenes in paternal/+. B shows that Dlx1/Dlx2 expression is much higher in Maternal/+, less in Paternal/+, and even less in WT when the transgene was inverted. C and D show chromosome compaction models for how the digit enhancers can interact with Dlx1/Dlx2.

 

Assignment -> limb development

Leave a reply

Part I – The study of limb phenotypes (10 min)

1.       What big processes of development are involved in the formation of a human limb?

The formation of three axes (AP, PD, and DV) is very important for limb development. The anterioposterior (AP) axis is formed first. These axes are caused by gradient differences in proteins like SHH and other molecules.

2.       Think about human limb development (wild-type or mutant) as a phenotype of interest. From a fundamental research perspective, why is it a useful phenotype to study? Why is it a good model system for the study of development? What are the advantages?

It is a good phenotype to study because it is very visual, caused by non-lethal mutations and changes in regulation, and it is a relatively complex model for even more complex systems. Limb malformation studies have been happening for a very long time even without genetic techniques.

3.       What is the difference between an isolated and a syndromic malformation, and what kinds of mutations are they postulated to be associated with?

isolated -> change in regulation of a gene in only one aspect of development. ie. SHH production is only effected in limb development but not everywhere else.

syndromic -> either a change in regulation in multiple areas or a mutation in the gene itself that causes phenotypic effects in many areas, leading to a group of symptoms that result in a syndrome

Part II – The study of cis-regulatory elements (20 min)

4.       Select one of the loci discussed in the review by Bhatia and Kleinjan. As a group, prepare a model of its regulation (can be in words, diagrams, a mixture thereof, etc). Then:

•   list the evidence that the authors use as a basis for each part of the model;

•   evaluate the evidence (decide if it is sufficient to support the various parts of the model);

•   if applicable, select a part of the model for which we do not (yet) have much supporting evidence. What additional piece(s) of evidence would help strengthen the model? What experiment(s) could you do to obtain them?

HoxD is surrounded by intergenic space and many regulatory islands that include enhancers. This includes the global control region (GCR) that has enhancing activity over many genes in a tissue specific manner; this is only one example of the many regulatory element groups surrounding HoxD. This information was found using histone markers that signify the presence of enhancers in the intergenic space surrounding HoxD. We think that finding histone makers that are often found near enhancers is a correlation rather than truly a signifier of enhancer sequences. Further investigation using enhancer traps and other techniques is necessary for verifying the presence of these regulatory islands.

5.       What is synteny? How does progress in our identification of cis-regulatory elements help explain some cases of synteny? (And thus making the connection between genome structure, function and evolution relevant?)

synteny -> physical co-localization of genes on the same chromosome

An understanding of the identity and location of cis-regulatory elements shows which aspects of regulation are essential for specific structure development. The conversation of these structures may lead to conservation of the regulation and groups of genes that are regulated by the same elements are more likely to remain together throughout speciation, resulting in syntenic regions.  

 Part III – Where do the cases are from, and who is the information for? (10 min)

6.       Think about all the research conducted on human limb malformation. How do you think the subjects for the study were recruited? How do you think the information gained from these studies was disseminated? Who had access to it? Who could it be useful or interesting for? How are the phenotypes under study depicted?

Subjects were likely recruited through doctors’ offices, medical histories, or speaking with people who have limb malformations. There may have also been some study of dead embryos (most likely in mice). The information gained from these studies was likely distributed through medical case studies, medical journals, and potentially in some scientific journals. Medical professionals, medical researchers and students, and other scientists would have had access to this information. This information would be interesting to medical students, science students, doctors, genetic counselors, researchers in the field, and people who are affected or know someone affected. The phenotypes are depicted in pictures, x-rays, and medical terms.

2nd final question attempt

Leave a reply

For my question I would like to look at something to do with how increased salinity affects amphibian development, I’m thinking something like “How does gene expression change in developing amphibians of species with varying salinity tolerances in different concentrations of salt?” and then I could look at gene expression using microarray with future applications looking at specific genes that might be down/upregulated.

Informal post -> science in the news

Leave a reply

http://news.sciencemag.org/brain-behavior/2015/04/reality-check-sex-crime-genetic

This article on Sciencemag.org was written in response to a study that was published looking at the potential for genetic connection to sex crimes committed by males in Sweden. They found that there was strong correlation between families and sex crimes. I found this interesting because of our class discussion/guest presentation on Apr 8, where we talked about how genetics is portrayed in the media/understood by the public. The article itself talks about how environment plays a large role in how children behave later in life and how genetics similar to a sex offender does not mean that you will commit a sex crime, but I can see how this could be taken in a different way where someone thinks that because their father has committed a sex crime then they will too. Definitely relevant to social psychology.

http://ije.oxfordjournals.org/content/early/2015/04/05/ije.dyv029.full

Informal Post -> lab personnel

Leave a reply

I’m currently doing my Honours project in a lab on campus and one of the research/lab tech’s contract is not being renewed for lack of funding. He has been in the lab for 11 years and is truly an incredibly helpful and easy to talk to person, who made coming into the lab  this summer painless, pretty fun, and a learning experience. His leaving reminded me of something I’ve thought about in the past. As a lab PI, a business owner, or lots of other positions, your support staff can make or break the feeling and atmosphere in your place of work. Competence is not the only aspect of a job that is important, but also the ability to welcome/help/interact with new people, as well as people who have been around for a long time. Surrounding yourself and your work with good, competent people can make a lot of difference, especially in a situations where people come and go like labs with grad students.