lecture 37-38 study guide

Recombinant human growth hormone (rhGH) and insulin are prepared commercially for medical use. In both cases, the genes for these proteins were cloned into plasmids, expressed in safe laboratory strains of the bacterium E. coli, purified, and given to patients. Many human proteins that would be useful in treating patients for a wide variety of diseases cannot be produced in this way. Why?

Additional steps—post-translational modifications—may be required before the protein can begin doing its particular job in the cell. Certain amino acids may be chemically modified by the attachment of sugars, lipids, phosphate groups, or other additions. Enzymes may remove one or more amino acids from the leading (amino) end of the polypeptide chain. In some cases, a polypeptide chain may be enzymatically cleaved into two or more pieces. In other cases, two or more polypeptides that are synthesized separately may come together, if the protein has quaternary structure; an example is hemoglobin (see Figure 5.18)

Describe the "genetic marker" that is identified by genome-wide association studies.

Among the most useful genetic markers in tracking down genes that contribute to diseases and disorders are single base-pair variations in the genomes of the human population. A single base-pair site where variation is found in at least 1% of the population is called a single nucleotide polymorphism (SNP, pronounced "snip"). Once a SNP is identified that is found in all affected people, researchers focus on that region and sequence it. In nearly all cases, the SNP itself does not contribute directly to the disease in question by altering the encoded protein; in fact, most SNPs are in noncoding regions. Instead, if the SNP and a disease-associated allele are close enough to be genetically linked, scientists can take advantage of the fact that crossing over between the marker and the gene is very unlikely during gamete formation. Therefore, the marker and gene will almost always be inherited together, even though the marker is not part of the gene (Figure 20.15). SNPs have been found that correlate with diabetes, heart disease, and several types of cancer, and the search is on for genes that might be involved.

How would genome-wide association studies be valuable for identifying genes associated with a specific disease?

An alternative approach is to analyze the genomes of large numbers of people with a certain phenotypic condition or disease, such as heart disease or diabetes, to try to find differences they all share compared with people without that condition. The assumption is that these differences may be associated with one or more malfunctioning genes, thus in a sense being naturally occurring gene knockouts. In these large-scale analyses, called genome-wide association studies, researchers look for genetic markers, DNA sequences that vary in the population.

Describe how the CRISPR-Cas9 system is being used as a biotechnological tool for plants, animals, and humans.

CRISPR-Cas9 is a unique technology that enables geneticists and medical researchers to edit parts of the genome? by removing, adding or altering sections of the DNA? sequence. It is currently the simplest, most versatile and precise method of genetic manipulation and is therefore causing a buzz in the science world.

What is a cloning vector?

Cloning vector = a DNA molecule that can carry foreign DNA into a host cell and replicate there. Bacterial plasmids are widely used as cloning vectors

You have ligated your PCR fragment (containing the gene that encodes your protein of interest) into an expression plasmid. How will you determine that the sequence of your gene is intact, contains no mutations, and will produce the desired protein?

Dideoxy chain termination method sequences nucleotides in any DNA fragment of up to 800-1000 base pairs in length

What is the advantage in using pluripotent embryonic stem (ES) cells compared to adult stem cells when generating different types of cells in culture?

Embryo stem cells are easier to grow than adult stem cells and can theoretically give rise to all types of cells in an organism. The range of cell types that can arise from adult stem cells is not yet fully understood

How would you determine that your recombinant plasmid contains a DNA fragment of the correct size?

Gel electrophoresis

How would you determine where a particular gene is expressed in an organism?

In situ hybridization which identifies the mRNAs being made by attaching a fluorescent label to a short single stranded nucleic acid called a nucleic acid probe RT PCR

An 18-year-old young lady has just delivered a healthy, 8 lb. baby girl. Upon questioning, she admits that there are three young men that could be the biological father. All have agreed to be tested for paternity. How would a laboratory perform the tests?

PCR is used to amplify particular STRs, using sets of primers that are labeled with different-colored fluorescent tags; the length of the region, and thus the number of repeats, can then be determined by electrophoresis. The PCR step allows use of this method even when the DNA is in poor condition or available only in minute quantities: A tissue sample containing as few as 20 cells can be sufficient.

What is a plasmid?

Plasmids = small circular DNA molecules that are replicated separately. A plasmid has only a small number of genes. These genes may be useful when the bacterium is in a particular environment but may not be required for survival or reproduction under most conditions

What is unique about the polymerase used in PCR?

Polymerase = Taq, an unusual heat stable DNA polymerase. It is found in the bacterial species Thermus aquaticus which lives in hot springs

You (yes, you) are interested in studying a bacterial protein. How would you obtain a DNA fragment that contains the gene that encodes the protein?

Polymerase chain reaction (PCR)

When does a plasmid become a recombinant DNA molecule?

Recombinant DNA = researchers obtain a plasmid and insert DNA from another source into it.

Why is mammalian reproductive cloning very difficult to achieve?

Researchers have uncovered some reasons for the low efficiency of cloning and the high incidence of abnormalities. In the nuclei of fully differentiated cells, a small subset of genes is turned on and expression of the rest of the genes is repressed. This regulation often is the result of epigenetic changes in chromatin, such as acetylation of histones or methylation of DNA (see Figure 18.7). During the nuclear transfer procedure, many of these changes must be reversed in the laterstage nucleus from a donor animal for genes to be expressed or repressed appropriately in early stages of development. Researchers have found that the DNA in cells from cloned embryos, like that of differentiated cells, often has more methyl groups than does the DNA in equivalent cells from normal embryos of the same species

What are induced pluripotent stem cells (iPS) and what are the genes that gave these cells their pluripotent character?

Researchers transform differentiated cells into a type of ES cell by using a retrovirus to introduce extra, cloned copies of 4 stem cell master regulatory genes which give them the pluripotent character

Describe the natural purpose of restriction enzymes in bacteria.

Restriction enzymes protect the bacterial cell by cutting up foreign DNA from other organisms or phages. Each restriction enzyme is very specific, recognizing a particular short DNA sequence (restriction site) and cutting both strands at precise points within the restriction site

How are restriction endonucleases (restriction enzymes) and DNA ligase used in gene cloning? Include the terms restriction site, and sticky ends in your answer.

Restriction enzymes recognize short sequences in the DNA and cuts them up into restriction fragments. Each restriction fragment has at least one single stranded end called the sticky end. The sticky end cam form H bonds with complementary sticky ends. These associations are made permanent by DNA ligase, and a clone is made.

What is a restriction site?

Restriction site = particular short DNA sequences that are recognized and cut by restriction enzymes

In question 8 (above) you described a technique for obtaining a protein-coding gene directly from a bacterial chromosome. A. Why would this approach not work in mammalian cells?

The presence of noncoding regions (introns) can make a eukaryotic gene very long and unwieldy and they can prevent the correct expression of the gene by bacterial cells which do not have RNA splicing machinery

What is therapeutic cloning and why is it considered controversial?

Therapeutic cloning = cloning to produce embryonic stem cells to treat disease. It is controversial because the cells come from embryos donated by patients undergoing fertility treatments

What is a VNTR or STR and what do these genetic markers represent?

These are tandemly repeated units of two- to five-nucleotide sequences in specific regions of the genome. The number of repeats present in these regions is highly variable from person to person (polymorphic), and even for a single individual, the two alleles of an STR may differ from each other. For example, one individual may have the sequence ACAT repeated 30 times at one genome locus and 15 times at the same locus on the other homolog, whereas another individual may have 18 repeats at this locus on each homolog.

How is a transgenic animal produced?

They can introduce a gene (or other DNA) from an animal into the genome of another individual, often of a different species. This individual is then called a transgenic animal. To do this, they first remove eggs from a female of the recipient species and fertilize them in vitro. Meanwhile, they have cloned the desired gene from the donor organism. They then inject the cloned DNA directly into the nuclei of the fertilized eggs. Some of the cells integrate the foreign DNA, the transgene, into their genome and are able to express the foreign gene. The engineered embryos that arise from these zygotes are then surgically implanted in a surrogate mother. If an embryo develops successfully, the result is a transgenic animal that expresses its new, "foreign" gene. Assuming that the introduced gene encodes a protein desired in large quantities, these transgenic animals can act as pharmaceutical "factories."

How does the polymerase chain reaction (PCR) generate a target sequence from a chromosome?

They heat the genomic DNA to separate the strands and cooled to allow hydrogen bonding of short single stranded DNA primers complementary to sequences on opposite strands at each end of the target sequence. Finally, a heat stable DNA polymerase extends the primers in the 5'-3' direction

What is the purpose of gene cloning?

To make many copies of a gene or to amplify a particular gene and to produce a protein product

How would you obtain a DNA fragment that contains only the coding region of a mammalian gene?

Using eukaryotic host cells for expressing a cloned eukaryotic gene. This is done through a process called electroporation = a brief electrical impulse is applied to a solution containing cells to create holes in the plasma membrane for DNA to enter

When does a bacterial cell become a recombinant bacterium?

When the recombinant DNA is returned back into the bacterial cell