WRN plasmids are digested with the restriction endonuclease cell one and MLU one and fragments are purified from a gel. These expression vectors digested with cell one to MLU one WN fragments are then ligated into the cell one to MLU one sites of the yeast vector. The multi copy plasmid also contains a TRP one selectable marker.
W Rrn constructs are transformed into yeast following standard protocols and transformants are selected on synthetic complete glucose media Lacking tryptophan. Individual transformants are then streaked onto synthetic complete glucose media lacking tryptophan and plates are incubated from the master plate. To analyze the slow growth phenotype restoration in WRN transformed SGS one top three double mutant strain transformants are streaked on glucose or galactose containing plates.
To analyze the effect of WRN expression on HU and MMS Sensitivity spotting is done to determine the cell cycle distribution of WRN expressing yeast cells. DAPI staining is performed. Expression of WRN in heterologous host is analyzed by Western Blood Analysis.
Hi, I'm Robert Broch from the section on DNA hela cases in the laboratory of molecular gerontology at the National Institute on Aging, one of the institutes of the National Institutes of Health. Hi, I'm Monica Agarwal, also from section on DNA Heli cases in the laboratory of molecular genetology at National Institutes on Aging. Understanding the roles of human DNA repair proteins and genetic pathways is a affordable challenge to many researchers.
Genetic studies in mammalian systems have been limited due to the lack of readily available tools, including mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. Today we will show you procedures involving yeast as a model system to investigate the molecular and cellular functions of A DNA unwinding enzyme known as a helicase in the prevention of premature aging. We use these procedures in our laboratory to genetically characterize the role of human burner gene product that is defective in DNA repair and maintenance of genomic stability in the premature aging disorder.
Werner Syndrome Werner is one of the five human req family heli cases in east. There is only one named sgs one to study the role of human Werner in replication stress response. We have examined the effect of Werner expression in east mutant background on various biological endpoints.
So Let's get, let's get started. Since we will be using yeast as a heterologous host, our first step will be to clone the human Werner gene or its variance into a yeast vector. Werner plasmids were digested with the restriction Endonuclease Sal one and MLU one and shall purified fragments within then ligated into the Sal one MLU one sites of the yeast expression vector that is under the regulation of a gallon inducible promoter.
The multi copy plasmid also contains a TRP one selectable marker. These Werner expression plasmid are then transformed into the appropriate tryptophan tropic yeast strains used in this study. And transformants are selected by plating the suspension on synthetic complete glucose media.
Lacking tryptophan incubate plates at 30 degrees Celsius for three to four days until the colonies appear. Once the colonies have grown streak, individual transformants on synthetic complete glucose media and incubate plates at 30 degrees Celsius for two to three days. The mutant ye strains expressing the human weer gene and its variants are then investigated for cellular phenotypes by the techniques demonstrated in the following sections.
The top three mutant shows a severe growth defect. However, mutation in the SGS one gene suppresses the slow growth phenotype of the top three mutant to examine the effect of Werner expression on the growth of wild type S Gs one or S Gs one top three strains streak the corresponding strains with the vector or Werner expression plasmid onto synthetic complete media plates lacking tryptophan but containing either 2%glucose or 2%gala lactose as a positive control. The effect of SGS one expression was determined incubate plates at 30 degrees for two days.
Wild type and SGS one mutant strain transformed with vector or Werner do not show any difference in their growth pattern. However, Werner transformed SGS one top three double mutant strain grow significantly slower than S GS one top three double mutant transformed with vector to assess the functional requirements of Warner to restore the slow growth phenotype. In the sgs one top three background streak sgs one top three strains transformed with Warner catalytic domain mutants onto synthetic complete plates lacking tryptophan containing either 2%glucose or 2%galactose after two days.
Incubation Werner Exonuclease mutant indicated as E 84 A restores the slow growth phenotype like Werner, thus showing that exonuclease activity is not required for top three slow growth phenotype restoration. Werner Helicase mutant grows well, thus demonstrating that Werner helicase activity is required for top three slow growth phenotype restoration. To determine the ability of Werner to affect the growth rate of sgs.
One top three strain at lower levels of protein expression streak sgs one top three strains transformed with the vector Werner or sgs one onto synthetic complete plates lacking tryptophan, but containing increasing concentrations of galactose. Incubate all plates at 30 degrees Celsius for two days. Warner and Warner Exonuclease mutant restore the top three slope with phenotype at all levels of galactose.
However, Warner Helicase mutants do not. It has been shown that the SGS one top three double mutant is less sensitive than the top three single mutant to the DNA alkylating agent methyl methane SULFONATE or MMS or the replication inhibitor hydroxyurea or HU to determine the effect of Warner expression on the MMS and HE sensitivity of SGS one or S GS one top three strains. Grow yeast strains transformed with vector or Werner in synthetic complete raffinose media lacking tryptophan at 30 degrees Celsius as a control.
Use a wild type parental strain transformed with vector re inoculate all cultures in synthetic complete raffinose media lacking trytophan and grow at 30 degrees Celsius to early log phase. When the cultures are at early log phase, prepare tenfold serial dilution up to 10, 000 fold of these strains using synthetic complete media lacking trytophan spot four microliters of each dilution onto synthetic complete GAL media lacking tryptophan plates containing 10 millimolar HU or 0.0025%MMS incubate the plates at 30 degrees Celsius for four days. An SGS one top three double mutant that is transformed with wild type sgs.
One is delayed in the late SG two phase of the cell cycle resulting in dumbbell shaped morphology. In contrast, wild type yeast complete cell division, leaving fewer dumbbell shaped cells to study the cell cycle distribution of the Werner transformed sgs one top three strain. First grow logarithmically growing cultures of the following strains in synthetic complete RAF media lacking tryptophan at 30 degrees Celsius overnight.
SGS one top three transformed with vector Werner or SGS one and the vector transformed Wildtype parental strain re inoculate the cultures at OD 0.05. Once re inoculated grow cultures to OD 0.5 and harvest by centrifugation Werner expression is then induced by adding gala lactose at 2%concentration. Allow the cultures to grow at 30 degrees Celsius for six hours.
After six hours, harvest the cultures, collect cells by centrifugation. Transfer the cultures to micro centrifuge tubes after centrifugation, wash the cells once with PBS. Then fix the cells in 70%ethanol for 20 minutes at room temperature after washing the fixed cells twice with PBS resuspend cells in one XPBS.
Place the cell suspension on a slide and add vector shield. Mounting media with DAPI at one microgram per milliliter concentration. Examine the slides under a XI Saxo vert microscope to look for cells with dumbbell shaped nuclei, which is characteristic of yeast cells in SG two phase of the cell cycle.
To determine expression of Werner protein or its variance in transformed SGS one or SGS one top three yeast strains. Start by growing the strains for six hours in galactose containing media to induce the expression of Werner protein. After collecting cells by centrifugation, wash them with PBS and resuspend.
An alkaline lysis buffer. Boil the cells for five minutes, then clarify by centrifugation and neutralize with one molar hydrochloric acid. These lysates are now ready for electrophoresis in eight to 16%poly acrylamide SDS gels to determine the level of expression of Warner protein under the influence of varying concentrations of galactose.
First, induce the cultures with varying galactose concentrations for six hours. Then harvest the cells by centrifugation and resuspend them in puffer containing protease inhibitors. Add glass beads 425 to 600 microns to the mixture and break the cells by vortexing vortex at high speed for a minute, followed by icing for a minute.
Repeat for a total of eight times. Next, centrifuge the resulting homogenate for five minutes at 15, 000 Gs and collect the supernatant fraction. After measuring the amount of protein in the lysate fractions by the Bradford Method, resolve equal concentrations of protein on eight to 16%Poly acrylamide SDS gels expression of Werner is then determined by Western blot Werner expression in sgs.
One top three restores the slow growth phenotype of top three as shown in these photographs. When sgs one top three strains transformed with vector Werner or sgs one streak on an SC tryptophan plate containing either 2%glucose or 2%galactose. The galactose induced expression of Werner causes the SGS one top three strain to grow more slowly.
In contrast, the wild type parental strain or SGS one strain transformed with the Werner is unaffected in its growth, indicating that the slow growth conferred by Werner is specific to the sgs. One top three background. When SGS one top three strains transformed with variants of Werner were streaked on SC tryptophan plates containing either 2%glucose or 2%galactose only.
The exonuclease dead Werner was still able to restore the slow growth phenotype. This demonstrates that Werner ATPase helicase but not exonuclease activity is required to restore the slow growth phenotype of top three. In an SGS one top three background, Werner restored the slow growth phenotype of SGS one top three throughout the GAL concentration range.
These results indicate that restoration of the slow growth phenotype in sgs one top three mutant background occurs at low Werner protein expression levels. When logarithmically growing cultures of sgs, one top three strains transformed with Werner or its variants were spotted in tenfold serial dilution onto SC trytophan plates containing glucose or galactose and either MMS or HU.We observed that Werner expression increased the MMS and two sensitivity of the SGS one top three strain. Furthermore, this effect on MMS and HU sensitivity in the SGS one top three background requires Werner ATP's helicase, but not exon clease activity.
Finally, Werner expression also restores the characteristic SG two delay of top three as indicated by the elevated population of large butted cells. In the sgs one top three mutant cells expressing Werner. In comparison with sgs, one top three cells transformed with vector cells were stained with Dabi.
To visualize the nuclei and the arrows indicate undivided nuclei Werner expression. In the transformed sgs, one top three cells was induced by adding indicated galactose concentrations and cells were harvested six hours after induction. Equal amounts of total cell lysate were loaded on eight to 16%poly acrylamide SDS gels followed by western blood detection using anti Warner antibody.
We have shown you how to investigate the cellular phenotypes associated with defined yeast mutant backgrounds to clarify the cellular and molecular functions of human learner gene mediated by its catalytic activities and protein interactions. Yeast can be used as a model organism to study the functions of the gene in defined pathways. When doing these procedures, it is important to select an appropriate yeast mutant background to discern the role of gene under investigation in defined pathways.
So that's it. Thanks for watching and good luck with your experiments.
