BIOL/CHEM 3361/3161  Problem Set Final  
Name: 
UTD ID: 
Lecture Section (circle one): 
Lin/Boyd (001) 
Gavva (002) 
Stelling/Dodani (003) 
Instructions: 
• Include your name and UTD ID on each page. 
• For full credit, all parts of each question must be answered with your own work and explanations.  
• All answers must be handwritten on this handout. No additional paper should be attached.  
• This is like the problem sets but will be graded for accuracy. 
• No late or electronic work will be accepted. 
• A stapled paper copy must be turned in by 5 PM on Tuesday May 10, 2022, in the drop box  
below on the third floor of Founders outside office 3.606.


1 BIOL/CHEM 3361/3161  Problem Set Final  
Name: 
UTD ID: 
1. The structure of Aspirin or acetylsalicylic acid is shown below. Ionization of Aspirin can 
influence the  pH of the stomach.  

It is absorbed into the blood through the cells lining the stomach and the small intestine. Absorption  
requires passage through the plasma membrane, the rate of which is determined by the polarity of  
the molecule: charged and highly polar molecules pass slowly, whereas neutral hydrophobic ones  
pass rapidly. The pH of the stomach after the meal is about 1.5, and the pH of the contents of 
small  intestine is about 6.  
a) Given that at equilibrium the pH of a 0.15 M solution of Aspirin is 2.16, what is the pKa of  
Aspirin? 
b) Using the pKa from (a), what is the ratio of deprotonated Aspirin (Asp-) to protonated Aspirin  
(HAsp) in the stomach? 


BIOL/CHEM 3361/3161  Problem Set Final  
Name: 
UTD ID: 
c) Is more Aspirin absorbed into the bloodstream from the stomach or small intestine? 
Clearly  justify your answer 
d) During a short distance run, the muscles produce a large amount of lactic acid 
(CH3CH(OH)  COOH, Ka = 1.38 X 10−4) from their glucose stores. Given this fact, 
why might hyperventilation  (breathing rapidly and deeply) before a run be useful? 

BIOL/CHEM 3361/3161  Problem Set Final  
Name: 
UTD ID: 
2. The following reaction has a ΔG°’ = -61.9 kJ/mol at 37 °C.  
Phosphoenolpyruvate (PEP) + H2O ⇌ Pyruvate + Inorganic Phosphate (Pi) 
a) Is the reaction favorable under standard biochemical conditions? Explain your 
answer. 
b) What is the equilibrium constant (Keq’) of this reaction at 37°C? 
In cells, the direct hydrolysis of PEP rarely occurs. Rather, the hydrolysis of 
PEP and the reverse hydrolysis of ATP are coupled.  
c) Write out the coupled reaction and determine its ΔG°’ at 37°C. The hydrolysis 
of ATP has a ΔG°’  of -30.5 kJ/mol.

BIOL/CHEM 3361/3161  Problem Set Final  
Name: 
UTD ID: 
d) At cellular concentrations of reactants and products at 37°C, the ΔG’ is -15 kJ/mol. 
If ADP and  ATP are at 0.5 mM and 0.3 mM respectively, what is the ratio of 
pyruvate to PEP? 
e) If 10-fold more PEP is added, what will be the resulting change in ΔG’?
    
BIOL/CHEM 3361/3161  Problem Set Final  
Name: 
UTD ID: 
3. Coronaviruses express a nucleocapsid protein that is needed for propagation, 
transcription, and  assembly of the virus. The nucleocapsid protein must be phosphorylated 
by a kinase in the host cell  to carry out these functions. One such kinase that has been 
recently reported is glycogen synthase  kinase 3 or GSK-3. The following is the 10-letter 
sequence of the nucleocapsid protein that is  recognized and phosphorylated by GSK-3: SSRGTSPARM. 
Note: pKa N-terminus = 9.3; pKa R = 12.5; pKa T = 13; pKa S = 13; pKa C-terminus = 4.3 a) 
What is the sequence of the peptide using the three-letter amino acid abbreviations?  b) 
Draw the chemical structure of the peptide when it is at pH 8. Assign charges and label 
the peptide  bonds.  
c) What is the pI of the peptide? Do not use an online resource to calculate this value. 
Show your  work to receive credit. 
BIOL/CHEM 3361/3161  Problem Set Final  
Name: 
UTD ID: 
d) Which amino acid(s) of the peptide are most plausible to be phosphorylated?  
e) Does the pI increase, decrease, or stay the same if all the possible amino acids you 
stated in you  answer for part (d) from the sequence are phosphorylated? Explain why. 
BIOL/CHEM 3361/3161  Problem Set Final  
Name: 
UTD ID: 
4. Glycogen synthase (GS) catalyzes the formation of a(1🡪4) glycosidic bonds in glycogen 
by catalyzing  the transfer of UDP-glucose to a glycogen particle where UDP is a byproduct. 
It is regulated by  allosteric modulators and phosphorylation coupled to hormones. Assume GS 
is a K-system enzyme.  Given this information, answer the following questions. 
a) Do you expect the binding of the substrate UDP-glucose to favor the R or T state of the 
enzyme or  neither? 
b) Draw a plot of the rate of GS versus UDP-glucose. Label the axes, define the half-maximal  
substrate binding (K0.5) point, and mark the curve with the label UDP-glucose.  
c) GSK-3 from Question 3 above phosphorylates GS, thus inhibiting the enzyme. When GS is  
phosphorylated, do you expect the R or T state of the enzyme or neither to be favored? 


d) On the same plot that you drew for part (b) now add a second curve indicating the effect 
of  phosphorylation. Define the half-maximal substrate binding (K0.5) point and mark the 
curve with the  label GS-P.


BIOL/CHEM 3361/3161  Problem Set Final  
Name: 
UTD ID: 
5. Natural and artificial sweeteners.  
a) Draw a Haworth projection of the following α-D-glucose, β-D-fructose, and the disaccharide 
from  these two monosaccharides which is called sucrose or α-D-glucopyranosyl-(1→2)-β-D 
fructofuranose. Clearly indicate the carbon where the α or β notation is derived from on the  
monosaccharides and the glycoside linkage in the disaccharide.  


b) Sucralose or better known as Splenda is the chlorinated form of sucrose, but it is not 
metabolized through glycolysis like sucrose which can be broken down into glucose and 
fructose. Provide a  reasonable explanation for this.


BIOL/CHEM 3361/3161  Problem Set Final  
Name: 
UTD ID: 
6. Glycogen synthase kinase 3 beta (GSK-3β) is a serine-threonine kinase known for its 
ability to regulate  glycogen synthesis. In addition, it has been reported that GSK-3β plays 
important roles in many  intracellular pathways and numerous pathologies, such as Alzheimer’s 
disease and cancer. Inhibitors  have been developed to modulate GSK-3β activity and used as 
therapies. The kinetics of the enzyme  (GSK-3β) with and without its inhibitor is shown in 
the plot below.  
B
A 

1/v
0  
(sec/μM)  
1/[S](1/μM) 

a) Which line indicate the condition with inhibitor, line A or line B?  
b) What is the type of this inhibitor? Explain.  
c) Determine Vmax and Km for this enzyme in the inhibited state. Be sure to include the units.    

BIOL/CHEM 3361/3161  Problem Set Final  
Name: 
UTD ID: 
7. An enzyme is known to obey the Michaelis-Menten kinetics with a Kcat of 30.0 s-1.  
a) At what substrate concentration would this enzyme with a Km of 0.015 M operate at one quarter 
of its maximum rate?  
b) Determine the fraction of Vmax that would be obtained when [S] = 2Km.  
c) What is the efficiency of this enzyme? 

BIOL/CHEM 3361/3161  
Problem Set Final  
Name: 
UTD ID: 
12
BIOL/CHEM 3361/3161  Problem Set Final  
Name: 
UTD ID: 
8. Using the Table provided on the previous page: 
a) Calculate the standard free energy change as a pair of electrons is transferred from succinate  
to molecular oxygen in the mitochondrial respiratory chain. Please show your equations and  work 
for full credit. 
Faraday Constant: 96485 J/V.mol; Gas Constant: 8.314 J/mol; ΔpH: 1.4; ΔΨ: 0.175 

b) Based on your answer in part (a), calculate the maximum number of protons that   could be  
pumped out of the matrix into the IMS as these electrons are passed to oxygen. Assume 37°C.  

c) At which site(s) are these protons pumped? 

BIOL/CHEM 3361/3161  Problem Set Final  
Name: 
UTD ID: 
9. Based on your knowledge of oxidative phosphorylation, answer the following questions:  
a) Suppose you synthesize an ATP synthase that was devoid of the γ (gamma) subunit. How  would 
the catalytic sites of the β subunits of such an enzyme compare to one another? Why?  What if 
only the c-terminus of the γ subunit is missing? 


b) Cells contain a protein called IF that binds tightly to the catalytic site of ATP synthase, 
inhibiting  its activity under certain circumstances. Can you think of any circumstances where 
such an  inhibitor might be useful within a cell? 

BIOL/CHEM 3361/3161  Problem Set Final  
Name: 
UTD ID: 
10. Knowing what you have learned about enzyme mechanisms in this course, answer the following 
questions: 
Acetylcholinesterase, which breaks down acetylcholine, a neurotransmitter, is a target for 
potential  drugs for Alzheimer’s Disease, as well as chemical warfare agents. It utilizes the 
same catalytic  mechanism of serine proteases that were discussed in lecture and has a pH 
optimum of 7.4 at 37°C.  One of its unique structural features is the deep gorge formed from 
mostly aromatic amino acids, at the  bottom of which lies the active site residues.  
a) In the image below, identify the three missing amino acids using the three-letter amino 
acid code.  
b) What is the mechanism of action of these three amino acids?


BIOL/CHEM 3361/3161  Problem Set Final  
Name: 
UTD ID: 
Alkaline proteases are hydrolytic enzymes that cleave proteins into smaller peptides and amino 
acids at basic pH and elevated temperatures. Because of these properties, enzymes such as these 
have been common additives to laundry detergents since the 1960s to help remove stains by 
degrading the protein. This biotechnological innovation has paved way for the incorporation 
of natural and engineered enzymes into a range application beyond laundry detergents that 
impact our everyday lives. 
Assume that alkaline protease A utilizes the same catalytic mechanism of serine proteases 
that were  discussed in lecture, but it operates at pH 9 or above at a temperature of 60°C 
or above. 
c) How do you think an enzyme could carry out this transformation at pH 9? Justify your 
explanation  considering the side chain pKa values in the catalytic site at pH 7.4 you 
described in part a. 
d) How do you think an enzyme could work at 60°C? Justify your explanation considering protein  
structure and intermolecular forces and drawing comparison to an enzyme that operates at 37 °C. 

BIOL/CHEM 3361/3161  Problem Set Final  
Name: 
UTD ID: 
Score sheet (do not fill out) but include in your printout that you turn in. 
Question Points received 
1 /8 
2 /10 
3 /10 
4 /8 
5 /8 
6 /10 
7 /6 
8 /9 
9 /8 
10 /11 
Total /88
                                        
                                    

                                        
                                        PRACTICAL TOPIC 2:  
Forward genetics, reverse genetics, and reporter genes 
Weeks 8-9 
Practical 2 is comprised of three sections: forward genetics, reverse genetics and  reporter
 genes. These are pillars of approaches to understanding the function of genes  and, as such, 
 are utilized in nearly all genetic systems to which they are applicable. 
LEARNING OBJECTIVE(s) for PRAC 2 
A few major goals of Practical 2 are to: 
1) Understand how forward genetics screens are performed in a variety of eukaryotic  
organisms and how they may be tailored to suit different life cycles. 
2) Be able to interpret gene expression patterns based on transcriptional and  translational 
reporter gene fusions and what can be inferred about gene function.  
3) Be familiar with various approaches to reverse genetics, and appreciate the strengths  
and weaknesses of the different techniques. 
Your report will consist of: 
— answers to questions 1-8 from Part A, including applicable crossing schemes for  mutagenesis 
analysis in questions 1-7 and a deduced genetic pathway for  question 8 
— answers to questions 1-7 from Part B, including annotated drawings/photos of the  
reporter gene expression patterns 
— answers to questions 1-7 from Part C, including a table/graph (C1) and a diagram (C5). 
Most other answers for sections B and C can be accomplished with a couple sentences
76 
BACKGROUND 
Forward genetic screens 
With the discovery by Hermann Muller that ionizing radiation induces mutations, geneticists  
realized that mutant organisms could be generated at will and systematically screened for 
phenotypes  of interest. Mutant phenotypes provide information on the function of the wild-type 
allele and insight into  biological processes. In an early example, in 1908 Archibald Garrod 
connected a hereditary condition,  alkaptonuria, with the lack of a specific biochemical 
activity, the metabolism of benzene rings in  homogentisic acid. He suggested that the wild-
type version of the gene encodes the enzyme responsible  for this biochemical activity. 
With the ability to generate mutations at will, geneticists began to employ  systematic genetic 
screens to dissect other biological processes, and the genetic bases for entire  biochemical 
pathways were elucidated.  
The design of genetic screens to identify genes involved in specific biological processes 
is limited  only by the imagination of the geneticist. An example is the research by Seymour
 Benzer that led to the  field of behavioral genetics in the 1970s. Benzer believed mutations 
 could be identified that specifically  affect behavioral processes, such as one you are using 
 now, the process of learning and memory. At  the time, behavior was thought by many to be too 
 complex to be dissected genetically. However, Chip  Quinn, a graduate student in Benzer’s lab, 
 built on previous ideas and designed an ingenious screen to  identify learning- and memory 
 deficient mutants in Drosophila. Wild-type flies could be taught that a  pulse of odor would 
 be followed by a shock; later, when the flies smelled the odor, they would take  evasive action.
  When Quinn and Benzer subjected a mutagenized population of Drosophila to this  genetic screen, 
  they identified mutant strains of flies that could perceive the odor but seemed unable to  
  associate the odor with the stimulus; either they did not learn or could not remember.  
Two mutant genes identified in the study, dunce and rutabaga, were later shown to encode  
proteins involved in the production or degradation of the small signaling molecule cyclic 
adenosine  monophosphate (cAMP). At the time, signaling via a cAMP pathway was known to be required 
for  learning in the sea hare, Aplysia. Since both Drosophila mutants were defective in cAMP physiology,  
other genes that encoded proteins involved in cAMP signaling and response were also investigated 
for  roles in learning. Ultimately, a transcription factor called creb (cAMP response element binding 
protein),  which activates or represses genes in response to cAMP signaling, was shown to be critical
 for storing  memories in flies. Remarkably, creb is widely conserved in animal species, and mouse 
 mutants lacking  creb activity also fail to remember. A similar gene is found in our genome.  
A great strength of forward genetic screens is that they are unbiased; no prior knowledge 
of the  molecular function of the encoded gene product is required. In a sense, by performing a 
mutagenesis,  the geneticist is allowing the organism to reveal how its biological processes operate.
 Following a  genetic screen, the number of genes and number of alleles of each gene identified are 
 determined by  complementation tests. For genes thought to act in the same pathway, the order 
 in which the genes act 
77 
can be determined by examining the phenotypes of double mutants. 
In this practical you will perform a modified version of Figure 16c. 
Evaluation of genetic pathways and epistasis 
Three distinct types of genetic pathways can be identified. Biosynthetic pathways are networks 
of interacting genes that produce a molecule or compound as their end product. Compounds such as  
pigments, amino acids, nucleotides, hormones, and so on are examples of the products of biosynthetic  
pathways. Signal transduction pathways are a second type of multiple-gene pathway. These pathways  
are responsible for reception of chemical signals, such as hormones, that are generated outside
 a cell  and initiate a response inside a cell. Signal transduction operates through the release 
 of a signaling  molecule that is part of a sequence of steps culminating in the activation or 
 repression of gene  expression in response to an intracellular or extracellular signal. Finally,
  developmental pathways  consist of genes that direct the growth, development, and differentiation 
  of body parts and structures.  Numerous developmental pathways have been identified in 
  organisms, and the functions of their genes  have been determined by experimental analyses of mutant phenotypes. 
Analysis of double mutants in genes acting in the same pathway can allow the ordering of the  genes 
within the pathway. Epistatic interactions, in which an allele of one gene modifies or prevents the  
expression of alleles at another gene, provide information of which of the two genes acts upstream
 and  which acts downstream of the other. In the case of biochemical pathways, epistasis analysis is usually  
 straightforward, as it is usually assumed that that the enzymes act in a linear pathway (with branched 
78 
or parallel pathways also possible) in which a substrate is sequentially converted through a series
 of  intermediates to a final product. Analysis of the intermediates present in the different 
 mutant alleles  provides substantial information as to the order in which the genes act. Furthermore, 
 the proteins  encoded by the genes in the pathway almost always act in a positive manner. In 
 contrast, in both signal  transduction and developmental pathways, the proteins encoded by 
 the genes can act either as positive  or negative regulators and there is often no way of 
 assaying intermediate steps in the developmental  pathway.  
Two approaches facilitate the ordering of genes in a developmental pathway. First, if both  
recessive loss-of-function and dominant gain-of-function alleles are available for some of the
 genes in  the pathway, epistatic interactions between such alleles can be powerfully informative. 
 Epistatic analysis  of developmental pathways, conducted by studying recessive and dominant 
 mutations of the same  gene, can be used to identify a group of genes that interact to control 
 a particular cellular process or  pathway and to establish an order-of-function map for the 
 genes in the pathway. The analysis of double  mutants and the availability of gain-of-function 
 alleles can be crucial in these endeavors. Alternatively,  if an assay to visualize gene 
 expression is available, examining the expression pattern of one gene in  the mutant background 
 of a second gene allows conclusion to be drawn about the relative order of action  of the two genes. 
Reporter genes 
A gene can act as a reporter if its product can be detected directly or is an enzyme that produces 
 a detectable product. The regulatory sequences of the gene of interest are used to drive the 
 expression  of the reporter gene. Two types of reporter gene fusions can be constructed: 
 transcriptional and  translational.  
In a transcriptional fusion, regulatory sequences directing transcription of the gene of interest
 are  fused directly with the coding sequences of the reporter gene. In this case, the reporter 
 gene will be  transcribed in the pattern directed by the regulatory sequences to which it is fused. 
 In translational  fusion, not only the regulatory sequences but also the coding sequence of the gene 
 of interest are fused  to the reporter gene in such a way that the reading frame for translation 
 is maintained between the gene  of interest and the reporter gene. As a result, the reporter 
 protein is translationally fused with the protein  of interest, and the location of the reporter 
 protein provides information not only on the spatial and  temporal transcriptional expression 
 pattern but also on the subcellular location of the fusion protein. In  translational fusions, 
 care must be taken to find out if the fusion protein is still functional, since the  addition of 
 the reporter protein could interfere with the proper folding or activity of the protein of interest.  
                                            
                                    

                                        
                                        Need to make a 2500 words report:
TOPICS:
• Health service market and their failures 
• Private health insurance market and their failures
• Analysis of spending on health services
• Applying economics on the field of health
• Production and costs in health services
• Physicians payment system
• Hospital funding system
• Need the news to be attached in separate documents
• Read all the instructions in the PDF properly 

                                    

                                        
                                        PRACTICAL TOPIC 2:  
Forward genetics, reverse genetics, and reporter genes 
Weeks 8-9 
Practical 2 is comprised of three sections: forward genetics, reverse genetics and  
reporter genes. These are pillars of approaches to understanding the function of genes  
and, as such, are utilized in nearly all genetic systems to which they are applicable. 
LEARNING OBJECTIVE(s) for PRAC 2 
A few major goals of Practical 2 are to: 
1) Understand how forward genetics screens are performed in a variety of eukaryotic  
organisms and how they may be tailored to suit different life cycles. 
2) Be able to interpret gene expression patterns based on transcriptional and  
translational reporter gene fusions and what can be inferred about gene function.  
3) Be familiar with various approaches to reverse genetics, and appreciate the strengths  
and weaknesses of the different techniques. 
Your report will consist of: 
— answers to questions 1-8 from Part A, including applicable crossing schemes for  
mutagenesis analysis in questions 1-7 and a deduced genetic pathway for  question 8 
— answers to questions 1-7 from Part B, including annotated drawings/photos of the  
reporter gene expression patterns 
— answers to questions 1-7 from Part C, including a table/graph (C1) and a diagram (C5). 
Most other answers for sections B and C can be accomplished with a couple sentences
76 
BACKGROUND 
Forward genetic screens 
With the discovery by Hermann Muller that ionizing radiation induces mutations, geneticists  
realized that mutant organisms could be generated at will and systematically screened for 
phenotypes  of interest. Mutant phenotypes provide information on the function of
 the wild-type allele and insight into  biological processes. In an early example, 
 in 1908 Archibald Garrod connected a hereditary condition,  alkaptonuria, with 
 the lack of a specific biochemical activity, the metabolism of benzene rings 
 in  homogentisic acid. He suggested that the wild-type version of the gene 
 encodes the enzyme responsible  for this biochemical activity. With the ability 
 to generate mutations at will, geneticists began to employ  systematic genetic 
 screens to dissect other biological processes, and the genetic bases for entire  
 biochemical pathways were elucidated.  
The design of genetic screens to identify genes involved in specific biologica
l processes is limited  only by the imagination of the geneticist. An example
 is the research by Seymour Benzer that led to the  field of behavioral genetics 
 in the 1970s. Benzer believed mutations could be identified that specifically  
 affect behavioral processes, such as one you are using now, the process of learning 
 and memory. At  the time, behavior was thought by many to be too complex to be 
 dissected genetically. However, Chip  Quinn, a graduate student in Benzer’s lab,
  built on previous ideas and designed an ingenious screen to  identify learning- 
  and memory deficient mutants in Drosophila. Wild-type flies could be taught that a 
   pulse of odor would be followed by a shock; later, when the flies smelled the odor, 
   they would take  evasive action. When Quinn and Benzer subjected a mutagenized 
   population of Drosophila to this  genetic screen, they identified mutant strains 
   of flies that could perceive the odor but seemed unable to  associate the odor 
   with the stimulus; either they did not learn or could not remember.  
Two mutant genes identified in the study, dunce and rutabaga, were later shown
 to encode  proteins involved in the production or degradation of the small 
 signaling molecule cyclic adenosine  monophosphate (cAMP). At the time, signaling 
 via a cAMP pathway was known to be required for  learning in the sea hare, Aplysia. 
 Since both Drosophila mutants were defective in cAMP physiology,  other genes that 
 encoded proteins involved in cAMP signaling and response were also investigated for  
 roles in learning. Ultimately, a transcription factor called creb (cAMP response 
 element binding protein),  which activates or represses genes in response to cAMP 
 signaling, was shown to be critical for storing  memories in flies. Remarkably, 
 creb is widely conserved in animal species, and mouse mutants lacking  creb 
 activity also fail to remember. A similar gene is found in our genome.  
A great strength of forward genetic screens is that they are unbiased; no prio
r knowledge of the  molecular function of the encoded gene product is 
required. In a sense, by performing a mutagenesis,  the geneticist is allowing 
the organism to reveal how its biological processes operate. Following a  
genetic screen, the number of genes and number of alleles of each gene 
identified are determined by  complementation tests. For genes thought to act in 
the same pathway, the order in which the genes act 
77 
can be determined by examining the phenotypes of double mutants. 
In this practical you will perform a modified version of Figure 16c. 
Evaluation of genetic pathways and epistasis 
Three distinct types of genetic pathways can be identified. Biosynthetic 
pathways are networks of interacting genes that produce a molecule or compound 
as their end product. Compounds such as  pigments, amino acids, nucleotides, 
hormones, and so on are examples of the products of biosynthetic  pathways. 
Signal transduction pathways are a second type of multiple-gene pathway. These 
pathways  are responsible for reception of chemical signals, such as hormones, 
that are generated outside a cell  and initiate a response inside a cell. Signal 
transduction operates through the release of a signaling  molecule that is part 
of a sequence of steps culminating in the activation or repression of gene  
expression in response to an intracellular or extracellular signal. Finally, 
developmental pathways  consist of genes that direct the growth, development, 
and differentiation of body parts and structures.  Numerous developmental pathways 
have been identified in organisms, and the functions of their genes  have been 
determined by experimental analyses of mutant phenotypes. 
Analysis of double mutants in genes acting in the same pathway can allow
 the ordering of the  genes within the pathway. Epistatic interactions, in which 
 an allele of one gene modifies or prevents the  expression of alleles at another 
 gene, provide information of which of the two genes acts upstream and  which acts 
 downstream of the other. In the case of biochemical pathways, epistasis analysis 
 is usually  straightforward, as it is usually assumed that that the enzymes act 
 in a linear pathway (with branched 
78 
or parallel pathways also possible) in which a substrate is sequentially 
converted through a series of  intermediates to a final product. Analysis of the 
intermediates present in the different mutant alleles  provides substantial information 
as to the order in which the genes act. Furthermore, the proteins  encoded by the 
genes in the pathway almost always act in a positive manner. In contrast, in both 

First, if both  recessive loss-of-function and dominant gain-of-function alleles 
are available for some of the genes in  the pathway, epistatic interactions between 
such alleles can be powerfully informative. Epistatic analysis  of developmental 
pathways, conducted by studying recessive and dominant mutations of the same  gene, 
can be used to identify a group of genes that interact to control a particular 
cellular process or  pathway and to establish an order-of-function map for the 
genes in the pathway. The analysis of double  mutants and the availability of 
gain-of-function alleles can be crucial in these endeavors. Alternatively,  if an 
assay to visualize gene expression is available, examining the expression pattern 
of one gene in  the mutant background of a second gene allows conclusion to be 
drawn about the relative order of action  of the two genes. 
Reporter genes 
A gene can act as a reporter if its product can be detected directly or is
 an enzyme that produces  a detectable product. The regulatory sequences of the 
 gene of interest are used to drive the expression  of the reporter gene. Two types 
 of reporter gene fusions can be constructed: transcriptional and  translational.  
In a transcriptional fusion, regulatory sequences directing transcription 
of the gene of interest are  fused directly with the coding sequences of the reporter 
gene. In this case, the reporter gene will be  transcribed in the pattern directed by 
the regulatory sequences to which it is fused. In translational  fusion, not only the 
regulatory sequences but also the coding sequence of the gene of interest are fused  
to the reporter gene in such a way that the reading frame for translation is maintained 
between the gene  of interest and the reporter gene. As a result, the reporter protein 
is translationally fused with the protein  of interest, and the location of the reporter 
protein provides information not only on the spatial and  temporal transcriptional 
expression pattern but also on the subcellular location of the fusion protein. 
In  translational fusions, care must be taken to find out if the fusion protein 
is still functional, since the  addition of the reporter protein could interfere 
with the proper folding or activity of the protein of interest.