Myofibers express a motor protein called myosin heavy chain (MHC). Focus on the bottom box in the center of the slide. MHC is the most abundant protein expressed in muscle. It is both a structural and regulatory protein that forms the backbone of the fiber (see next slide), and also drives the contraction process of the fiber. Slow motor units contain fibers that express what is termed a slow, type I MHC, which intrinsically causes the fiber, when activated electrically by the nerve, to contract slowly.

In contrast, the other two motor units express faster types of MHC, called isoforms (i.e., different forms of a nearly identical protein), and are designated as either IIa, IIx, or Iib, depending on the individual properties of the motor units. It is important to note that in the faster units, many of the fibers express two or more types of MHC. We don’t know why this is the case, but it is postulated that this characteristic enables the fibers to change their contractile properties quickly (over a period of a few days), depending on the physiological conditions imposed on the muscle.

Can you think of a type of activity that may serve as a stimulus to induce expression of the slower myosin isoforms? What might induce faster isoforms?

Suggested Reading:
Caiozzo, V. J., Baker, M. J., Huang, K., Chou, H., Wu, Y. Z., & Baldwin, K.M. (2003). Single fiber myosin heavy chain polymorphism: How many patterns and what proportions? Am J Physiol Regul Integr Comp Physiol. 285: R570-R580.

ADDITIONAL NOTES FROM SPEAKER’S TRANSCRIPT (http://www.bioedonline.org/presentations/).
Look at the middle of the slide, and focus first of all on the fibers that are designated in yellow. These fibers express a certain motor protein that we call myosin heavy chain (MHC). Think of myosin heavy chain as the motor that drives the contraction process. The unique feature of these fibers is that they express only one kind of a motor protein, which we refer to as slow or type I myosin. The green and red fibers in the middle are the ones that the individual was using in the last slide, while cycling the bicycle. This motor unit, or this grouping of fibers, consists of two faster types of myosin (isoforms called IIa and IIx), one that is expressed as green, and another expressed as red. If we move to the right, we see another type of fiber that is really fast. These fibers express the fast myosin heavy chain and allow the muscle to shorten with a much more explosive capability, as illustrated on the right with the individual executing the burst power. Myosin heavy chains have evolved and been conserved over millions of years, and through many species. While I show these fibers to you in the human body, just about every animal system has the same types of myosin.

In addition to muscle size, the contractile phenotype (type of myosin expressed) can change under different conditions of loading and unloading. In the normal muscle on the left, the fibers express primarily the slow type I MHC isoform and some of the fast IIa isoform. Following prolonged exposure to microgravity models, the muscle becomes atrophied and at the same time, expresses predominantly fast MHC isoforms, especially the type IIx, which imparts faster contractile properties on the muscle. Thus, in this state the muscle would be compromised by the combined loss in size, strength and ability to perform activities that are heavily dependent on continually overcoming the force of gravity.

How would you predict the muscle performance capacity/capability of an individual returning to Earth from long-term space flight with these defined muscle properties?

Suggested Reading:
Baldwin, K.M. & Haddad, F. (2002). Skeletal muscle plasticity: cellular and molecular responses to altered physical activity paradigms. Am. J. Physical Med. & Rehab. 81: (Suppl) S40-S51.
Caiozzo, V.J., Baker, M.J., Huang, K., Chou, H., Wu, Y.Z., & Baldwin, K. M. (2003). Single fiber myosin heavy chain polymorphism: How many patterns and what proportions? Am J Physiol Regul Integr Comp Physiol. 285: R570-R580.

ADDITIONAL NOTES FROM SPEAKER’S TRANSCRIPT (http://www.bioedonline.org/presentations/)
When we unload the muscles under the conditions described, the muscle fibers get smaller. If we look at the type of myosin expressed in this particular muscle, it expresses mostly type I myosin heavy chain. Over time, as the muscle gets smaller, it also changes its phenotype, and it expresses 2-X and 2-B types of fibers that are fast, creating a normal-sized muscle that atrophies, and the phenotype is switched to a faster type. What do you think would be the consequence of shifting this muscle to a smaller, faster size? Do you think muscles under these conditions would be very effective in supporting gravity?

Many genes have been discovered by an approach called linkage analysis. Linkage analysis relies on Mendelian inheritance of chromosomes (and genes). In linkage analysis, polymorphic genetic markers (sites of DNA sequence variations) spread throughout the genome are used to track the transmission of various genomic regions through a family. With linkage analysis, all members of a family are genotyped for the polymorphic markers. The genotype of each family member at each marker is then compared to the genotypes of other family members to determine which markers are present in the family members affected by the disorder. Since linkage analysis does not detect mutations in genes directly, but rather traces a potential disease allele through a family because of a tight association (genetic linkage) between the potential disease gene and the marker, further investigation is required once a genomic region of interest is identified. The goal of the investigation is to identify the gene and mutations associated with the disorder. Since linkage analysis is not a direct mutation detection method, its level of sensitivity relies on how many different forms of the markers there are, the density of the markers throughout the genome, and the genetic distance between the marker and the disease gene. Highly polymorphic markers, and lots of them, improve the success rate of linkage analysis.

Linkage analyses must be performed with data sets based on large, multigenerational families. This is because one needs many individuals, and typically more than one affected individual, within a family to identify the genomic regions associated with a disorder with statistical significance. However, large families segregating genetic disorders are not always easy to find, especially in highly mobile societies like the United States. In some cases, a several small families can be used instead, but this approach requires that the disease is caused by the same gene in all the families. For example, if the disorder is caused by mutations in the same gene in every individual affected by the disorder, then the results of linkage analysis of many small families can be added together because everyone affected by the disorder should share the same disease gene. One example of such a disorder is cystic fibrosis (CF), in which all individuals affected by the disorder have mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). On the other hand, if a disorder is caused by mutations in more than one gene, linkage analysis will be more successful if large, multigenerational families are used because two different families might have the disease because of mutations in different genes. In these cases, the linkage data would appear contradictory and would not be additive. One example of a highly heterogeneous genetic disorder is nonsyndromic hearing loss. More than 100 genes have been mapped so far for hereditary deafness.

Genetic heterogeneity has complicated the search for the genetic factors associated with a number of common complex disorders such as schizophrenia and autism. In these disorders, multiple genetic factors are likely to be involved and each may have only a small effect on susceptibility to develop the disorder. As such, the disorder in any two families may be associated with different sets of genes. Sorting out the genetic factors involved is quite complicated and requires extensive additional investigations.

As a result of the Human Genome Project, researchers now have a collection of polymorphic genetic markers that provide excellent coverage of the genome. These markers are powerful tools for mapping disease genes. Linkage analysis strategies are precise, accurate and effective, but labor intensive. However, while they often require data from large families, they do not require an investigator to know anything about the underlying biochemical mechanisms involved in the disorder.

In the simplified diagram on this slide, a polymorphism associated with an autosomal recessive disorder is indicated by the red star. Only the affected individual has inherited two copies of the disease-associated polymorphism. From this finding, researchers would explore the genetic region surrounding the marker to identify genetic alterations unique to the disease-associated form of the marker.

Because of the specificity of their DNA sequence recognition, restriction enzymes can also be used to detect mutations associated with genetic diseases. In the image shown in this slide, the restriction enzyme DdeI is used to identify the mutation in the beta-globin gene that is associated with sickle cell disease in a fragment of DNA amplified from patients by polymerase chain reaction (PCR). In this case, the mutation associated with sickle cell disease changes the DNA sequence of the beta-globin gene in such a way that it is no longer recognized by DdeI (Panel A). As a result, normal, non-sickle, beta-globin genes are digested by DdeI while sickle cell-associated alleles are not (Panel B).

When DNA from patients is amplified by PCR and the PCR products are digested with DdeI and run on agarose gel electrophoresis, a person with two normal beta-globin genes will demonstrate a two band pattern, indicating that the beta-globin DNA was cut by DdeI (Panel C, Row 1). A person with two copies of the sickle cell-associated gene will show only an uncut band, indicating that neither of the beta-globin genes was cut by DdeI (Panel C, Row 2). A carrier for sickle cell disease will demonstrate both cut and uncut bands (Panel C, Row 3), indicating that he or she has one normal gene and one sickle cell-associated gene.

Understanding the molecular genetic basis of a disorder and developing a DNA-based methodology for mutation detection enable precise DNA-based diagnosis of the disorder, permitting identification of affected and unaffected individuals, and carriers of recessive diseases. In this example, the individual in row 2 of Panel C is affected by sickle cell disease, while the individual in row 3 is a carrier for sickle cell disease.

Southern analysis permits the detection of mutations and polymorphisms in genes because changes in the sequence of genes result in changes in restriction patterns. In Southern analysis, genomic DNA is isolated from a patient’s cells and digested with an enzyme. The resulting DNA fragments are run on an agarose gel for separation according to size. To stabilize the DNA in its electrophoretic separation pattern, the DNA is hybridized (affixed) to a nylon membrane. A labeled DNA probe (short sequence of DNA tagged isotopically or chemically) specific for the gene of interest is mixed with the membrane under conditions that allow the probe to find and bind to (hybridize with) its complementary DNA on the membrane, resulting in a characteristic banding pattern for the normal gene upon isotopic or chemical signal detection. This approach can be used to analyze a particular nucleotide change if the change occurs at the site of action by a restriction enzyme and alters the ability of the enzyme to cut the DNA. This approach also can detect alterations in genes that disrupt the regular banding pattern because of deletions or other rearrangements in the gene that span restriction sites. Southern analysis also can be effective in identifying changes in the size of repetitive elements, such as triplet repeats that are associated with disease. In many cases, the changes in size of the gene associated with expansion of the repeat sequence is large enough to be seen on Southern analysis as a change in the size of the band associated with the gene. In cases of smaller repeat expansions a different type of gel substance, called polyacrylamide, is used because it is better able to separate smaller sized DNA fragments.

In the image on the slide, genomic DNA is digested with a restriction enzyme and separated by gel electrophoresis according to fragment size. The DNA is then transferred from the gel to a nylon membrane and hybridized with a radioactively labeled probe unique to the sequence of interest. In this case, the probe is complementary to the beta-globin gene and is designed to detect the mutation associated with sickle cell disease, which interrupts a restriction enzyme site. The sickle cell-associated genes remain undigested by the enzyme while the normal gene sequence is cut by the enzyme into a smaller piece of DNA. In the lane labeled A, an individual with two normal copies of the beta-globin gene was analyzed. The size of the fragment seen on the autoradiograph is that of the digested DNA, indicating two normal alleles and no sickle cell-associated allele. In the column labeled S, an individual with sickle cell disease and two copies of the sickle cell gene was analyzed. The size of the fragment seen is larger than the fragment in column A, indicating that the restriction enzyme failed to cut the DNA due to the presence of two copies of the sickle cell-associated mutation. In the column labeled AS, an individual with one copy of the normal gene and one copy of the sickle cell-associated gene was analyzed. Two bands are seen: one for the digested smaller allele on the bottom; and, one for the uncut sickle cell-associated allele on the top. This individual is a carrier for sickle cell disease.

The polymerase chain reaction (PCR) was invented in 1985 by Kary Mullis, PhD. Dr. Mullis received the Nobel Prize in Chemistry in 1993 for his work.

PCR is the enzymatic amplification of DNA, creating millions of copies of a segment of DNA from only a small amount of starting material. To begin, DNA primers are manufactured that are complementary to a portion of the target DNA sequence. The primers and the target DNA are mixed and denatured (separated into single strands) by high temperature. The mixture is then cooled, allowing the primers to hybridize (bind) to the target DNA. After hybridization, a special DNA polymerase enzyme that is resistant to heat inactivation synthesizes new DNA strands from the primers, using the target DNA as a template. The process is repeated for 30-50 cycles of heat denaturation, primer binding, and DNA synthesis. After many cycles of PCR, millions of copies of the target DNA are created where only a few existed before. PCR is also fast. Most PCR reactions take only a few hours to perform in the laboratory.

DNA fragments generated by PCR can be subjected to a variety of different analyses, depending on the type of mutation or polymorphism sought. For example, gel-based methods that separate fragments based on size are ideal for detecting small expansions or deletions such as occur in repetitive elements, such as trinucleotide repeats, while DNA sequencing will detect changes, such as single nucleotide substitutions, in the sequence of a gene.

PCR has several advantages over methods like Southern analysis. First, PCR uses very small amounts of DNA as starting material, while Southern analysis and other methods require large amounts of input DNA. PCR also can use degraded DNA as starting material because even a degraded DNA sample (DNA that is broken into pieces by decay) is likely to have pieces still large enough to be bound by the primers and amplified while Southern analysis tyipcally examines much larger pieces of DNA. These properties make PCR useful for forensic DNA analyses where evidentiary DNA is frequently scarce and/or compromised by environmental or other exposures.

In the early days of PCR, the methodology could only amplify DNA fragments a few hundred to two or three thousand base pairs in length. Recent improvements in PCR technology allow today’s scientists to amplify DNA fragments that are up to several thousand base pairs in length, but even today PCR cannot amplify very long target DNA sequences.

ASO (allele specific oligonucleotide) hybridization begins with PCR. Once amplified, the target DNA is hybridized (affixed) to a nylon membrane or another solid substrate in spots (without prior electrophoresis). Oligonucleotide probes specific for normal and mutant sequences are radioactively, chemiluminescently, or fluorescently labeled and hybridized to the substrate-bound target DNA. Competitive binding between mutant and normal probes permits probe binding dependent upon the sequence of the target DNA.

ASO is very effective and reliable and is used for many DNA tests where only one or a few mutations cause the disease. The development of 96- and 384- tray formats permit automation and the simultaneous analysis of many specimens, making ASO useful for high throughput diagnostic testing. ASO effectively identifies sought after mutations, but unlike sequencing, cannot typically detect mutations not directly tested. Another common problem with ASO is with polymorphic sequences that occur near the sequence variation that is being sought. If these polymorphisms interfere with probe binding, false positive or false negative results can be obtained. In these cases, special consideration must be given to probe design to avoid errors in interpretation of ASO test results.

The image on the slide shows the ASO analysis of a mutation in the CFTR gene associated with cystic fibrosis (CF) in a family with one affected child and a fetus of unknown genotype. In this example, the parents are both carriers of a CF-associated allele. On ASO analysis, they show a signal with both the normal and mutant probes, consistent with their carrier status. The child, who is affected by CF, shows an ASO signal only for the mutant probe, indicating inheritance of two copies of the CF allele, one from each parent. The fetus also has inherited a CF-associated allele from each parent and does not show a signal from the normal probe. This fetus is predicted to be affected with CF. The column labeled X represents the negative control.

There are a number of variations on allele specific methods, all based on the same principle: primers and probes only bind to complementary DNA sequences. This feature of DNA hybridization is exploited to identify normal and mutant alleles in patient specimens.

Missense mutations result in the substitution of one amino acid for another in the sequence of a protein. Missense mutations can interrupt the functional capabilities of proteins in which they occur, or they may have no effect at all on a protein's function. Missense mutations that do not have pathological consequences can become frequent in the population because they are not selected against. Such mutations are typically called polymorphisms to distinguish their benign nature from that of pathologically significant mutations.

A polymorphism is a DNA sequence variation that is common in the population (as opposed to a mutation, which is more rare). The term "polymorphism" also is used frequently in clinical genetics to distinguish a DNA sequence variation that is unrelated to disease (polymorphism) from a DNA sequence variation that is associated with a disease (mutation). A polymorphism occurs in the human genome roughly every 250-1,000 base pairs.

Polymorphisms provide markers, or signposts, throughout the genome that can be used to trace traits and diseases, and their associated genes, through families or large populations. This experimental approach has allowed researchers to map the genetic locations of numerous disease genes, facilitating their identification. The polymorphism itself may not necessarily be associated with the disease in question, but it may sit on the chromosome very near the genetic alteration associated with the disease, and thus, may be used as a tag for the disease gene until a more precise genetic alteration associated with the disease can be identified.

The slide shows an electropherogram generated by automated fluorescent DNA sequencing of a segment of DNA using an ABI 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The arrow shows a nucleotide substitution in one of the two copies of this gene in this individual. One copy of the gene (allele) carries an A nucleotide (green peak) while the other allele carries a C nucleotide (blue peak). This nucleotide substitution is detected as a dual peak in the output of the sequence analysis software. In this case, the DNA sequence variation results in a missense mutation. Missense mutations can be associated with disease or they may be benign polymorphisms. The detection of variations in the DNA sequence of a gene that are uncertain biological consequence can complicate the interpretation of genetic test results.

The human genome also contains a number of repetitive elements or tandemly repeated sequences. The size of these repeated sequences varies among individuals. Many of these variable length repetitive sequences are benign and simply represent normal variability in the human genome, while some are associated with disease. The polymorphic nature of repetitive DNA sequences is the basis for much of the DNA-based forensic analyses performed today including paternity and personal identification testing.

As a species, humans are more than 99.9% identical at the level of our DNA. However, with 3 billion base pairs in our haploid genome, this leaves a lot of room for variation. Thus, each of us is unique, with the exception of identical twins who share the same genetic makeup.

Human genetic polymorphisms have been used recently to trace the history of our species. What has been learned is that humans evolved in Africa about 100,000 years ago. Although the dates are only broad estimations based on calculated mutation rates, we now know that we all are descended from a small group of perhaps 10,000-80,000 individuals. Over time, humans migrated out of Africa in several waves and eventually populated the globe. From these studies, we have learned that every human alive today is related to every other: we are all descended from the same trunk of the human family tree, brothers and sisters separated only by time and geography.