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Thread: Questions Concerning Myostatin

  1. #1 Questions Concerning Myostatin 
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    All of this info comes from a 1997 article in Genome Research:

    Mutations in myostatin (GDF8) in Double-Muscled Belgian Blue and Piedmontese Cattle
    Ravi Kambadur1,2, Mridula Sharma1, Timothy P.L. Smith3, and John J. Bass


    A recent study demonstrated that mice lacking a normal copy of the myostatin gene (GDF8) display a phenotype with significant similarities to the double muscling seen in cattle. myostatinis a member of the transforming growth factor β (TGF-β) gene superfamily specifically expressed in skeletal muscle of adult mice, as well as during early development. In the mouse study, the third exon of the gene was replaced with a neo cassette, removing the portion of the protein that is highly conserved among the TGF-β superfamily of genes.
    What are neo cassettes? What are other important TGF-β members?

    Belgian Blue cattle are homozygous for an 11-bp deletion in the coding region that is not detected in cDNA of any normal animals examined.
    In English please...


    The relative increase in fiber number is observed early in pregnancy (Swatland and Kieffer 1974) and results in a calf possessing nearly twice the number of muscle fibers at the time of birth. The occurrence of double muscling has been observed in several cattle breeds worldwide since it was first documented by Culley in 1807.
    Awesome.

    However, problems associated with the trait, such as reduction in stress tolerance, fertility, and calf viability in Belgian Blue have hindered exploitation of the hypertrophy by classical genetic selection (Arthur 1995).
    What causes these negative side effects?


    Figure 1.
    Sequence analysis of normal- and double-muscled bovine myostatin. Sequencing was performed on three independent normal- and double-muscled alleles, and one representative sequence of both alleles is shown. (A) Amino acid sequence comparisons of mouse (MMYO.PRO) and bovine Myostatin (BMYO.PRO) proteins. Nonconserved amino acids are indicated by solid bars. The consensus amino acid sequence is shown at the top. The proteolytic processing site is underlined. (B) The deletion mutation is detected by fluorometric sequencing of myostatin cDNA from normal- and double-muscled cattle. The sequence of the double-muscled allele is shown above that of the normal allele (Control), and the position where 11 bp is deleted in the mutant allele is indicated by an arrow. The large bracket in the normal allele sequence denotes the region that is deleted in the double-muscled allele. (C) The amino acid sequence of Myostatin in normal cattle is shown above the predicted amino acid sequence of Myostatin in double-muscled cattle. The premature stop codon at amino acid position 288 in the double-muscle allele is indicated by an asterisk (*). (D) The predicted amino acid sequence of Myostatin in normal cattle is shown below that of the Piedmontese breed in the vicinity of the transition mutation. The altered residue in the Piedmontese allele is underlined. Asterisks (*) indicate two of the nine conserved cysteine residues in exon 3 of the normal bmyostatin allele.


    Figure 2.
    (A,B) Agarose gel electrophoresis of PCR products (513 bp) obtained from RT–PCR using total RNA from embryos or fetuses of different normal (A)- or double (B)-muscled Belgian Blue bovine developmental stages. (M) Markers (1-kb ladder from GIBCO BRL). Different embryonic or fetal ages are indicated in corresponding lanes. The locations of the primers used to amplify 513-bp partial cDNA are from amino acid 202 to 208 (sense primer) and from amino acid 365 to 371 (antisense primer). (See Methods for primer sequence.) This 513-bp partial cDNA contains the 11-bp deletion observed in double-muscled Belgian Blue cattle. (C) Expression of myostatin in different adult bovine muscles. Fifteen micrograms of total RNA was electrophoresed on formaldehyde–agarose gel, blotted onto nylon membrane, and probed with bovine myostatin cDNA. (Lane 1) M. gastrocnemius; (lane 2) M. psoas major; (lane 3) M. longissimus dorsi; (lane 4) M. biceps femoris; (lane 5) M. diaphragm; (lane 6) M. semimembranosus; (lane 7) M. flexor digitorum longus; (lane 8) M. vastus medialis; (lane 9) M. vastus lateralis; (lane 10) heart; (lane 11) M. cutaneus trunci; (lane 12) M. semitendinosus; (lane 13) M. semitendinosus (normal, 260 day); (lane 14) M. semitendinosus (double-muscle, 260 day).
    How is a sequence analysis done? What is RT–PCR? Please explain how to read these results...


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  3. #2  
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    No takers?


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    gottspieler, a lot of this you can look up for yourself.

    http://en.wikipedia.org/wiki/Gene_cassette

    http://en.wikipedia.org/wiki/Transfo...ta_superfamily

    http://en.wikipedia.org/wiki/Zygosity

    http://en.wikipedia.org/wiki/Coding_region

    http://en.wikipedia.org/wiki/CDNA
    (cDNA is made from RNA transcripts, so if you can't find the cDNA of a certain gene or allele in an animal, that means that gene is not being expressed or the animal doesn't have that allele.)

    What causes these negative side effects?
    Your best bet is to look up the paper that the author references as support for that statement. But it may not be known. Many genes have affects on many different biochemical pathways, and when one thing goes wrong, the effects can be widespread and various. If nothing else, growing and maintaining muscles consumes lots of energy, and that energy may be siphoned away from that which is usually devoted to the maintenance of other systems.

    http://en.wikipedia.org/wiki/Protein_sequencing

    http://en.wikipedia.org/wiki/Real-ti...chain_reaction
    (RT-PCR is typically used to detect the expression level of a certain gene.)

    It would help best to actually see the figures, but I'd imagine the first figure is just showing the results of the sequencing of protein from normal and mutant animals - basically showing you where the mutation is. It seems that the second figure is showing the levels of expression of the gene in normal and mutant animals from different muscles and/or ages. I have no idea what that's telling you without a picture or a description of the results.
    Man can will nothing unless he has first understood that he must count on no one but himself; that he is alone, abandoned on earth in the midst of his infinite responsibilities, without help, with no other aim than the one he sets himself, with no other destiny than the one he forges for himself on this earth.
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    gottspieler, a lot of this you can look up for yourself.
    I was feeling lazy. Thanks for explaining the RT-PCR.
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  6. #5  
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    Quote Originally Posted by gottspieler
    gottspieler, a lot of this you can look up for yourself.
    I was feeling lazy. Thanks for explaining the RT-PCR.
    Next time don't be lazy! We're not here to do people's work for them. Just like with students coming for homework "help."
    Man can will nothing unless he has first understood that he must count on no one but himself; that he is alone, abandoned on earth in the midst of his infinite responsibilities, without help, with no other aim than the one he sets himself, with no other destiny than the one he forges for himself on this earth.
    ~Jean-Paul Sartre
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    Sure thing. :wink:
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