Download CHAPTER 4 Gene Control of Proteins

Peter J. Russell
CHAPTER 4
Gene Control of Proteins
edited by Yue-Wen Wang Ph. D.
Dept. of Agronomy,台大農藝系
NTU
遺傳學 601 20000
Chapter 4 slide 1
Gene Control of Enzyme Structure
• 1. Genes encode proteins, including enzymes.
• 2. Genes work in sets to accomplish biochemical
pathways.
• 3. Genes often work in cooperation with other
genes.
• 4. These discoveries are the foundation of
modern molecular genetics.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 2
Garrod’s Hypothesis of Inborn Errors of
Metabolism
•
1. Alkaptonuria is a human trait characterized by urine blackening on exposure to air
and arthritis in later life.
•
2. Archibald Garrod and William Bateson (1902) concluded alkaptonuria is
genetically determined because:
• a. Families with alkaptonuria often had several affected members.
• b. Alkaptonuria is much more common in 1st cousin marriages than marriages with
unrelated partners.
•
3.Garrod showed that alkaptonuria results from homogentisic acid (HA) in the urine.
HA is absent from normal urine. Garrod reasoned that normal people metabolize
HA, but those with alkaptonuria do not because they lack the necessary enzyme. He
termed this an inborn error of metabolism (Figure 4.1).
•
4. The responsible mutation is recessive. The gene was later shown to be on
chromosome 3.
•
5. Garrod’s work was the 1st evidence of a specific relationship between genes and
enzymes. With the insight that a mutation can block a human metabolic pathway by
damaging an enzyme, causing a detectable buildup of that enzyme’s substrate, he
found a similar relationship in three other human diseases. His work, naturally, was
not appreciated by his contemporaries.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 3
Fig. 4.1 Phenylalanine-tyrosine metabolic pathways
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 4
The One Gene-One Enzyme Hypothesis
• 1. Genes act by regulating definite chemical events.
• 2. George Beadle and Edward Tatum (1942) showed a
direct relationship between genes and enzymes in the
haploid fungus Neurospora crassa. This led to their one
gene-one enzyme hypothesis, and a share of the 1958
Nobel Prize in Physiology or Medicine.
• Animation: The One Gene-One Enzyme Hypothesis
台大農藝系 遺傳學 601 20000
Chapter 4 slide 5
• 3. It is necessary to understand the life cycle of Neurospora crassa
(orange bread mold) to understand Beadle and Tatum’s work (Figure 4.2).
• a. Neurospora is a mycelial-form fungus with asexual spores. The spores
are called conidia. They are orange in color.
• b. It is a haploid organism, so mutations are easily spotted.
• c. Its life cycle is conveniently short.
• d. Neurospora propagates asexually by dispersal of:
•
i. bits of mycelium.
•
ii. conidia.
• e. It also propagates sexually by means of two mating types, A and a.
•
i. The two types are indistinguishable, except that A will not mate with
A, nor a with a.
•
ii. Only an A x a cross will result in gamete fusion, producing an A/a
diploid nucleus that quickly undergoes meiosis to produce four haploid
nuclei.
•
iii. After a round of mitosis, the ascus contains eight sexually produced
ascospores, each capable of forming a mycelium. Of the ascospores, four
are A and four are a.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 6
Fig. 4.2 Life cycle of the haploid, mycelial-form fungus Neurospora crassa
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 7
• f. Wild-type Neurospora needs only simple minimal media with:
•
i. inorganic salts (including nitrogen source).
•
ii. an organic carbon source (such as glucose or sucrose).
•
iii. biotin (a vitamin).
• g. To grow on minimal media, wild-type Neurospora synthesizes all
organic molecules it needs for growth. An auxotrophic mutant unable to
make a needed nutrient will only grow if that nutrient is provided as a
supplement in its medium.
• 4. Beadle and Tatum isolated auxotrophic mutants by mutating with X
rays and then crossing with a wild-type strain. The cross insured that
effects were due to inheritance, rather than direct damage from the
radiation. In their experiment:
• a. One progeny spore per ascus was germinated in a complete medium
so that growth would occur regardless of nutritional mutations. Then
growth was transferred to minimal media, where auxotrophs won’t
grow.
• b. Each mutant was then tested on an array of minimal media, each
with a different single supplement, to determine the type of nutritional
mutation (Figure 4.3).
台大農藝系 遺傳學 601 20000
Chapter 4 slide 8
Fig. 4.3 Method devised by Beadle and Tatum to isolate auxotrophic mutations in
Neurospora
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 9
• 5. Beadle and Tatum assumed that many genes interact in
Neurospora cells. They reasoned that metabolism
proceeded by series of reactions, each catalyzed by an
enzyme, and organized into pathways. The analysis of
methionine biosynthesis is an example of the analytical
approach they used (Table 4.1):
• a. Starting with a set of methionine auxotrophs, it was found that
4 genes are involved, met-2+, met-3+, met-5+, met-8+.
• b. Checked each mutant on series of minimal media each
supplemented with a different chemical believed to be involved
in the pathway. Expected growth if providing a chemical used
after the metabolic block, so the earlier the mutated gene
functions in the pathway, the more supplements will support
growth.
• c. Deduced the pathway of methionine synthesis, and correlated
mutations with enzymes used in the pathway.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 10
台大農藝系 遺傳學 601 20000
Chapter 4 slide 11
Fig. 4.4 Methionine biosynthetic pathway showing four genes in Neurospora crassa
that code for the enzymes that catalyze each reaction
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 12
• 6. Beadle and Tatum’s famous conclusion from
this type of experiment is that one gene encodes
one enzyme. Later work showed that some
proteins consist of more than one polypeptide,
and that not all proteins are enzymes. The
principle is now usually stated, “one gene-one
polypeptide.”
• iActivity: Pathways to Inherited Enzyme
Deficiencies
台大農藝系 遺傳學 601 20000
Chapter 4 slide 13
Genetically Based Enzyme Deficiencies in
Humans
• 1. Single gene mutations
are responsible for many
human genetic diseases.
Some mutations create a
simple phenotype, while
others are pleiotropic
(Table 4.2).
台大農藝系 遺傳學 601 20000
Chapter 4 slide 14
Phenylketonuria
•
http://www.genetic-counseling.com.tw/datebase/PKU_10.21.htm
1. Phenylketonuria (PKU) is commonly caused by a mutation on chromosome 12 in the
phenylalanine hydrolase gene, preventing the conversion of phenylalanine into tyrosine
(Figure 4.1).
•
2. Phenylalanine is an essential amino acid, but excess is harmful, and so is normally
converted to tyrosine. Excess phenylalanine affects the CNS, causing mental retardation, slow
growth and early death.
•
3. PKU’s effect is pleiotropic. Some symptoms result from excess phenylalanine. Others result
from inability to make tyrosine; these include fair skin and blue eyes (even with brown-eye
genes) and low adrenaline levels.
•
4. Diet is used to manage PKU by providing just enough phenylalanine for protein synthesis,
but not enough that it accumulates. To be effective, the special diet must commence in the first
two months after birth, continue at least throughout childhood, and be resumed before
pregnancy in PKU women to avoid phenylalanine levels that would affect the fetus.
•
5. All U.S. newborns are screened for PKU using the Guthrie test:
•
•
a. A drop of blood on filter paper is placed on solid media containing β-2-thienylalanine and the
bacterium Bacillus subtilis.
•
b. Normally, β-2-thienylalanine inhibits growth of Bacillus subtilis.
•
c. Phenylalanine allows Bacillus subtilis to grow in the presence of β-2-thienylalanine, so bacterial
growth indicates high phenylalanine levels in the blood, and the possibility that the infant has PKU.
6. NutraSweet is aspartame, which breaks down to aspartic acid and phenylalanine, with
serious consequences for a phenylketonuric.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 15
Albinism
• 1. Classic albinism results from an autosomal recessive
mutation in the gene for tyrosinase. Tyrosinase is used to
convert tyrosine to DOPA in the melanin pathway.
Without melanin, individuals have white skin and hair,
and red eyes due to lack of pigmentation in the iris.
• 2. Two other forms of albinism are known, resulting from
defects in other genes in the melanin pathway. A cross
between parents with different forms of albinism can
produce normal children.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 16
Lesch-Nyhan Syndrome
•
http://www.genetic-counseling.com.tw/datebase/Lesch_Nyhan_syndrome_10.30.htm
1. Lesch-Nyhan syndrome results from a recessive mutation on the X chromosome, in the gene
for hypoxanthine-guanine phosphoribosyl transferase (HGPRT). The fatal disease is found in
males, while heterozygous (carrier) females may show symptoms when lyonization of the normal
X chromosome leaves the X chromosome with the defective HGPRT gene in control of cells.
•
2. HGPRT is an enzyme essential to purine utilization. In Lesch-Nyhan syndrome this pathway is
highly impaired. Purines accumulate and are converted to uric acid.
•
3. Symptoms of Lesch-Nyhan syndrome:
•
•
a. Infants develop normally for several months. Orange uric acid crystals in diapers (of males) are only
clue of disease.
•
b. At 3–8 months, motor development delays lead to weak muscles.
•
c. Muscle tone is altered, producing uncontrollable movements and involuntary spasms.
•
d. At 2–3 years children show bizarre activity, such as compulsive self-mutilation that is difficult to
control and painful, as well as aggression toward others.
•
e. Lesch-Nyhan individuals score severely retarded on intelligence tests, possibly due to poor
communications skills.
•
f. Most Lesch-Nyhan individuals die before their 20s, typically from infection, kidney failure or uremia.
4. In the case of Lesch-Nyhan syndrome, a defect in a single enzyme, HGPRT, has very
pleiotropic effects, giving rise to uremia, kidney failure, mental deficiency and (so far
inexplicably) self-mutilation.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 17
Tay-Sachs Disease
http://www.genetic-counseling.com.tw/datebase/Tay-Sachs%20disease-11.20.htm
•
1. Tay-Sachs is one of a group of diseases called lysosomal-storage diseases.
Generally caused by recessive mutations, these diseases result from mutations in
genes encoding lysosomal enzymes.
•
2. Tay-Sachs disease (aka infantile amaurotic idiocy) results from a recessive
mutation in the gene hexA, which encodes the enzyme N-acetylhexosaminidase A.
The HexA enzyme cleaves a terminal N-acetylgalactosamine group from a brain
ganglioside.(Fig. 4.5)
•
3. Infants homozygous recessive for this gene will have nonfunctional HexA
enzyme. Unprocessed ganglioside accumulates in brain cells, and causes various
clinical symptoms:
• a. Infants have enhanced reaction to sharp sounds.
• b. A cherry-colored spot surrounded by a white halo may be visible on the retina.
• c. Rapid neurological degeneration begins about one year of age, as brain loses control of
normal functions due to accumulation of unprocessed ganglioside.
• d. Progress is rapid, with blindness, hearing loss and serious feeding problems leading to
immobility by age 2.
• e. Death often occurs at 3–4 years of age, often from respiratory infection.
•
4. The disease is incurable. Carriers and affected individuals can be detected by
台大農藝系 遺傳學 601 20000
Chapter 4 slide 18
genetic testing.
Fig. 4.5 The biochemical step for the conversion of the brain ganglioside GM2 to the
ganglioside GM3, catalyzed by the enzyme N-acetylhexosaminidase A (hex A)
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 19
Gene Control of Protein Structure
• 1. Genes also make proteins that are not enzymes.
Structural proteins, such as hemoglobin, are often
abundant, making them easier to isolate and
purify.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 20
Sickle-Cell Anemia
• 1. J. Herrick (1910) first described sickle-cell anemia, finding that red
blood cells (RBCs) change shape (form a sickle) under low O2 tension.
• a. Sickled RBCs are fragile, hence the anemia.
• b. They are less flexible than normal RBCs, and form blocks in
capillaries, resulting in tissue damage downstream.
• c. Effects are pleiotropic, including damage to extremities, heart, lungs,
brain, kidneys, GI tract, muscles and joints. Results include heart
failure, pneumonia, paralysis, kidney failure, abdominal pain and
rheumatism.
• d. Heterozygous individuals have sickle-cell trait, a much milder form
of the disease.
• 2. E.A. Beet and J.V. Neel independently proposed (1949) that sicklecell trait and disease were the result of a single mutant allele.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 21
• Animation: Gene Control of Protein Structure and
Function
• 3. Linus Pauling and coworkers (1949) used
electrophoresis and showed:
• a. Hemoglobin from individuals with sickle-cell anemia (Hb-S)
has altered mobility compared with normal hemoglobin (Hb-A).
• b. Hemoglobin from individuals with the sickle-cell trait shows
equal amounts of Hb-A and Hb-S, indicating that heterozygotes
make both forms of hemoglobin.
• c. Therefore, the sickle-cell mutation changes the form of its
corresponding protein, and protein structure is controlled by
genes.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 22
Fig. 4.7 Electrophoresis of hemoglobin variants
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 23
• 4. Hemoglobin is formed by four polypeptide
chains, two molecules of the α polypeptide and 2
of the β polypeptide, each associated with a heme
group (Figure 4.8).
台大農藝系 遺傳學 601 20000
Chapter 4 slide 24
Fig. 4.8 The hemoglobin molecule
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 25
• 5. V.M. Ingram (1956) found that the 6th amino
acid of the β chain in sickle-cell hemoglobin is
valine (no electrical charge) rather than the
negatively charged glutamic acid in the β chain of
normal hemoglobin (Figure 4.9).
台大農藝系 遺傳學 601 20000
Chapter 4 slide 26
Fig. 4.9 The first seven N-terminal amino acids in normal and sickled hemoglobin 
polypeptides
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 27
• 6. Outline of the genetics and gene products involved in
sickle-cell anemia and trait:
• a. Wild-type β chain allele is βA, which is codominant
with βS.
• b. Hemoglobin of βA/βA individuals has normal β
subunits, while hemoglobin of those with the genotype
βS/βS has β subunits that sickle at low O2 tension.
• c. Hemoglobin of βA/βS individuals is 1⁄2 normal, and 1⁄2
sickling form. (The two β chains of an individual
hemoglobin molecule will be of the same type, rather than
mixed.) These heterozygotes may experience sickle-cell
symptoms after a sharp drop in the oxygen content of
their environment.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 28
Other Hemoglobin Mutants
• 1. Screening of hemoglobin for altered
electrophoretic mobility has identified over 200
hemoglobin mutants, showing a variety of amino
acid substitutions in both the α and the β chains.
Each appears to derive from a single amino acid
change.
• 2. Most effects are not as severe as those seen in
sickle-cell anemia.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 29
Fig. 4.10a Examples of amino acid substitutions found in  polypeptides of various
human hemoglobin variants
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 30
Fig. 4.10b Examples of amino acid substitutions found in  polypeptides of various
human hemoglobin variants
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 31
Biochemical Genetics of the Human ABO Blood
Group
• 1. Cellular antigens are important in blood transfusions, since recipient
antibodies may responsd to antigens on donor cells.
• 2. Karl Landsteiner discovered human ABO blood groups in early
1900s and received 1930 Nobel Prize in Physiology and Medicine for
this work. Properties of the human ABO blood group (Table 4.3).
• a. There are three alleles at the ABO locus, IA, IB, and i. From these
three alleles, four phenotypes are produced:
• i. Type A individuals have the A antigen on their RBCs, and anti-B
antibodies in their blood. Their genotype is IA/IA or IA/i.
• ii. Type B individuals have the B antigen on their RBCs, and anti-A
antibodies in their blood. Their genotype is IB/IB or IB/i.
• iii. Type AB individuals have both the A and the B antigen on their
RBCs, and neither anti-A nor anti-B antibodies in their blood. Their
genotype is IA/IB.
• iv. Type O individuals have neither the A nor the B antigen on their
RBCs, and both anti-A and anti-B antibodies in their blood. Their
genotype is i/i.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 32
• 3. Anitgen-antibody relationships, and their
impact in blood transfusions, are summarized in
Fig. 4.11
台大農藝系 遺傳學 601 20000
Chapter 4 slide 33
Fig. 4.11 Antigenic reactions that characterize the human ABO blood types
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 34
• 4. Summary of the relationship between the ABO alleles and RBC
antigens:
• a. The ABO locus produces RBC antigens by encoding
glycosyltransferases which add sugars to existing polysaccharide on
membrane glycolipid molecules. These polysaccharides act as the antigen
in the ABO system.
• b. In most people, the glycolipid is the H antigen.
• i. Activity of the IA gene product, α-N-acetylgalactosamyl transferase,
converts the H antigen to the A antigen.
• ii. Activity of the IB gene product, α-D-galactosyltransferase, converts
the H antigen to the B antigen.
• iii. Both enzymes are present in an IA/IB individual, and some H
antigens will be modified to the A antigen while others are modified
to the B antigen.
• iv. Neither enzyme is present in an i/i individual, and so the H antigen
remains unmodified.
• 5. Production of the H antigen is controlled by a different genetic locus
from the ABO enzymes. Rarely, an individual lacks the dominant allele H
needed for H antigen production. This h/h genotype results in the
Bombay blood type, which is similar to type O except that Bombay blood
type individuals produce anti-O antibodies
that are遺傳學
not seen
in trueChapter
type 4 slide 35
台大農藝系
601 20000
O individuals.
Fig. 4.12 Human ABO blood type antigens
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 36
Cystic Fibrosis (囊腫纖維症)
• 1. Cystic fibrosis (CF) affects the pancreas, lungs and digestive system,
and sometimes the vas deferens in males. The disease is characterized
by abnormally viscous secreted mucus, and lung complications are
managed by percussion and antibiotics to treat infections. Life
expectancy with current treatments is about 40 years.
• 2. The affected gene is on the long arm of chromosome 7, and encodes
a protein called cystic fibrosis transmembrane conductance regulator
(CFTR). Comparing DNA sequences of cloned gene from normal and
CF individuals shows that the CF mutation commonly is the deletion of
a specific 3-bp region, removing one amino acid from the protein
product.
• 3. The structure of the protein has been deduced from its sequence
(Figure 4.13). CFTR has homology with a large family of active
transport membrane proteins.
• 4. Functional analysis shows that CFTR normally forms a chloride
channel in the cell membrane. The mutated gene results in an abnormal
CFTR protein, preventing chlorine ion transport and resulting in CF
symptoms.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 37
Fig. 4.13 Proposed structure for cystic fibrosis transmembrane conductance regulator
(CFTR)
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 38
Genetic Counseling
• 1. Genetic testing can detect many inherited enzyme and protein defects,
yielding information about whether an individual has a disease or is a carrier.
Chromosomal abnormalities can also be detected.
• 2. Genetic counseling is advice based on genetic analysis, focusing either on
the probability that an individual has a genetic defect, or the probability that
prospective parents will produce a child with a genetic defect. Genetic
counselors have the task of explaining diseases, probabilities and options to
affected individuals or parents.
• 3. Some aspects of human heredity are well understood, others not yet so
well. Effective genetic counseling requires up-to-the minute knowledge of
genetic research, and the ability to offer clients unbiased and nonprescriptive
information from two main sources:
• a. Pedigree analysis is an important tool of genetic counseling, considering
phenotypes found in both families over several generations. This is
particularly useful for identifying suspected carriers.
• b. Fetal analysis includes assays for enzyme activity or protein level, or
detection of changes in the DNA itself.
• 4. For most defective alleles, there is currently no way to change the
resulting phenotype, and so genetic counseling focuses primarily on
informing clients of risks and probabilities.台大農藝系 遺傳學 601 20000 Chapter 4 slide 39
Carrier Detection
• 1. A carrier is heterozygous for a recessive gene
mutation. In a cross between two carrier parents,
1⁄4 of the offspring are expected to develop the
disease, and 1⁄2 to also be carriers.
• 2. The carrier’s phenotype is normal, but if levels
of the affected protein are determined, they may
be well below those of a normal individual.
• 3. Detection of carriers by direct DNA testing is
discussed in Chapter 8.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 40
Fetal Analysis
• 1. Genetic counseling is also concerned with whether a
fetus is normal. A sample of fetal cells is needed for the
analysis. There are currently two methods of obtaining the
necessary sample:
• a. Amniocentesis is removal of a sample of amniotic fluid using
a syringe needle inserted through the uterine wall (Figure 4.14).
The procedure is seldom done before the 12th week of
pregnancy, due to small amounts of amniotic fluid and risk to the
fetus.
• b. Chorionic villus sampling can be done in the 8th–12th weeks
of pregnancy, by removal of chorionic villus tissue either
through the abdomen as in amniocentesis, or via the vagina
(Figure 4.15)
台大農藝系 遺傳學 601 20000
Chapter 4 slide 41
Fig. 4.14 Amniocentesis, a procedure used for prenatal diagnosis of genetic defects
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 42
Fig. 4.15 Chorionic villus sampling, a procedure used for early prenatal diagnosis of
genetic defects
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 43
• 2. Once fetal cells are obtained they are usually cultured
in the laboratory, although chorionic villus sampling may
provide enough tissue to assay directly. They are examined
for protein or enzyme alterations or deficiencies, DNA
changes and chromosomal abnormalities.
• 3. Amniocentesis is costly and cannot be performed until
the second trimester, removing early abortion as an option
in cases of severe genetic defects. Chorionic villus
sampling can be done earlier, but carries a higher risk of
fetal death and inaccurate diagnosis due to the presence of
maternal cells.
台大農藝系 遺傳學 601 20000
Chapter 4 slide 44