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38
CHAPTER 6
b.
Anti-Sm antibodies. The Smith antigen is a protein that keeps DNA in its cor-
rect shape.
c.
Anti-SS-A (Ro) antibodies. The Sjogren syndrome A antigen is a protein asso-
ciated with small RNAs called scYRNAs which are present in both the cyto-
plasm and nucleus.
d.
Anti-snRNP antibodies. Small nuclear ribonucleoproteins that are used in
RNA splicing were first characterized by using antibodies from SLE patients.
4.
Clinical findings include
autoimmune hemolytic anemia, thrombocytopenia,
leukopenia, lymphadenopathy, small joint inflammation, malar butterfly rash, dif-
fuse proliferative glomerulonephritis, pericarditis, Libman-Sacks endocarditis, lung
interstitial fibrosis, and pregnancy-related complete heart block in newborns
caused by anti–SS-A (Ro) antibodies crossing the placenta.
B.
-THALASSEMIA
1.
-Thalassemia is an autosomal recessive genetic disorder caused by 200 missense
or frameshift mutations in the HBB gene on chromosome 11p15.5 for the
-globin
subunit of hemoglobin.
2.
-Thalassemia is defined by the absence or reduced synthesis of -globin subunits
of hemoglobin.
a.
A
0
mutation refers to a mutation that causes the absence of
-globin sub-
units. The
-globin gene has three exons and two introns. The introns con-
tain a G–T sequence that are responsible for correct RNA splicing to occur
and form normal
-globin mRNA. In
0
-thalassemia, the G–T sequence is mu-
tated to A–T such that correct RNA splicing does NOT occur and the defec-
tive
-globin mRNA cannot be translated into -globin protein.
0
-thalassemia
produces severe anemia in the affected individuals.
b.
A
mutation refers to a mutation that causes the reduced synthesis of
-
globin subunits. It should be noted that the clinical amount of
-globin sub-
units of hemoglobin is due to two (2) alleles.
3.
The mutations in the HBB gene result in the reduced amounts of HbA (Hb
2
2
)
because there is reduced synthesis of
-globin subunits which are found only in HbA.
4.
Heterozygote carriers of
-thalassemia are often referred to as having thalassemia
minor.
a.
Thalassemia minor results from the inheritance of a
mutation of one
-
globin allele (
/normal
).
b.
Clinical features include individuals who are asymptomatic with very mild or
absent anemia, but red blood cell abnormalities may be seen.
5.
Prevalence.
The prevalence of
-thalassemia is very high in the African, Mediter-
ranean, Arabic, Indian, and Southeast Asian populations. The prevalence of
-
thalassemia is 1/7 births in Cyprus and 1/8 births in Sardinia.
6.
There are two clinically significant forms of
-thalassemia:
a.
Thalassemia major. Thalassemia major results from the inheritance of a
0
mu-
tation of both
-globin alleles (
0
/
0
) and is the most severe form of
-tha-
lassemia. An excess of
-globin subunits form insoluble inclusion bodies within
mature red blood cell precursors. Clinical features include microcytic hypochro-
matic hemolytic anemia, abnormal peripheral blood smear with nucleated red
blood cells, reduced amounts of HbA, severe anemia, hepatosplenomegaly, fail
to thrive, become progressively pale, regular blood transfusion are necessary, and
usually come to medical attention between 6 months S 2 years of age.
b.
Thalassemia intermedia. Thalassemia intermedia results from the inheritance
of a
0
mutation of one
-globin allele (
0
/normal
) and is a less severe form
of
-thalassemia. Clinical features include a mild hemolytic anemia, individ-
uals are at risk for iron overload, regular blood transfusions are rarely neces-
sary, and usually come to medical attention by
2 years of age.
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39
A. HOUSEKEEPING GENES. All cells in the human body contain housekeeping genes
which are expressed and produce housekeeping proteins that are used for many func-
tions common to all cells (e.g., enzymes for metabolic processes, cytoskeleton proteins,
and proteins essential to the endoplasmic reticulum and Golgi).
B. However, differentiated cells produce specialized proteins (e.g., hepatocytes produce
Factor VIII and the pancreatic beta cells produce insulin).
C. Because all cells in the human body contain identical DNA, the key cell biological ques-
tion is why do hepatocytes produce Factor VIII and not insulin? Or, why do pancreatic
beta cells produce insulin and not Factor VIII? The answers to these questions fall into
the area of gene expression or gene regulation.
Mechanism of Gene Expression (Figure 7-1).
The mechanism of gene expression
employs the use of both cis-acting DNA sequences and trans-acting proteins.
A. CIS-ACTING DNA SEQUENCES. The cis-acting DNA sequences are named “cis” be-
cause they affect the expression of only linked genes on the same chromosome. The
cis-acting DNA sequences act as binding sites for various trans-acting proteins. There
are a number of cis-acting DNA sequences which include the following:
1.
Core promoter sequence.
The core promoter sequence (e.g., TATA box se-
quence) is usually located near the gene (close to the initiation site where tran-
scription actually begins) and upstream of the gene. The core promoter is the site
where RNA polymerase II and TFIIs assemble to form the transcription-initiation
(TI) complex so that a gene may be transcribed into an RNA transcript. However,
the TI complex will produce only a basal level of transcription or constitutive ex-
pression.
2.
Proximal promoter region sequence.
The proximal promoter region sequence
(e.g., GC box sequence) located immediately upstream of the core promoter
sequence.
3.
Enhancer sequences.
The enhancer sequences are usually located far away from
the gene and either upstream or downstream of the gene. The enhancer sequences
increase the basal level of transcription or constitutive expression produced by the
TI complex.
4.
Silencer sequences.
The silencer sequences can be located near the core pro-
moter sequence, upstream of the core promoter sequence, or within introns. The
silencer sequences decrease transcription levels.
5.
Insulator sequences (boundary elements).
The insulator sequences are regions
of DNA that block (or insulate) the influence of enhancer sequences or silencer
sequences.
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40
CHAPTER 7
6.
Response element sequences.
The response element sequences are a short dis-
tance upstream of the core promoter sequence. The response element sequences
modulate transcription levels in response to external stimuli such as cAMP, serum
growth factor, interferon-
, heavy metals, phorbol esters, heat shock, or steroid
hormones. There are a number of response element sequences which include the
following:
a.
cAMP response element (CRE)
b.
Serum growth factor response element (SRE)
c.
Interferon-
response element (IRE)
d.
Heavy metal response element (HMRE)
e.
Phorbol ester response element (PRE)
f.
Heat shock response element (HSRE)
g.
Glucocorticoid response element (GRE)
B. TRANS-ACTING PROTEINS. The trans-acting proteins are named “trans” because
they effect the expression of genes on other chromosomes and migrate to their site of
action. There are two types of trans-acting proteins called transcription factors and
gene regulatory proteins. The trans-acting proteins bind to cis-acting DNA sequences.
1.
Transcription factors.
The general transcription factors (TFIIA, TFIIB, TFII D
TFIIE, TFIIF, and TFIIH) along with RNA polymerase II form the TI complex.
2.
Gene regulatory proteins.
The gene regulatory proteins bind to specific enhancer
sequences, silencer sequences, or insulator sequences and promote the production
of specialized proteins within a cell (e.g., hepatocytes produce Factor VIII and the
pancreatic beta cells produce insulin).
3.
Other trans-acting factors.
a.
CREB (cAMP response element binding protein) binds to CRE in response
to elevated cAMP levels in the cell caused by a protein hormone binding to a
G protein–linked receptor and thereby induces gene expression.
b.
Serum response factor binds to SRE in response to serum growth factor and
thereby induces gene expression.
c.
Stat-1 binds to IRE in response to interferon-
and thereby induces gene ex-
pression.
d.
Mep-1 binds to HMRE in response to heavy metals and thereby induces gene
expression.
e.
AP1 binds to PRE in response to phorbol esters and thereby induces gene ex-
pression.
f.
hsp70 binds to HSE in response to heat shock and thereby induces gene ex-
pression.
g.
Steroid hormone receptor binds to GRE in response to steroid hormones and
thereby induces gene expression.
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41
CONTROL OF GENE EXPRESSION
Proximal promoter
region
Upstream
Core promoter
TFIIs
TI complex
GRP
Other
GRP
GRP
Gene
E
n h
a
n
c
e
r
S
i
l e n ce
r
I
n
s
u
l a
t
o
r
R
e
sp
o
n
s
e
e
l e
m
e n
t
Start of
transcription
● Figure 7-1 Mechanisms of Gene Expression. The cis-acting DNA sequences include the core promoter sequence,
proximal promoter region sequence, enhancer sequences, silencer sequences, insulator sequences, and response ele-
ment sequences. These cis-acting DNA sequences act as binding sites for various trans-acting proteins. The trans-acting
proteins include the general transcription factors (TFIIs), gene regulatory proteins, and other trans-acting proteins.
The binding of the cis-acting DNA sequences and the trans-acting proteins modulate the activity of the transcription-
initiation (TI) complex. DNA looping allows trans-acting proteins that are bound at distant sites to interact with the TI
complex. GRP
gene regulatory protein; TFIIs general transcription factors for RNA polymerase II; TI transcription-
initiation; TATA
TATA box.
The Structure of DNA-Binding Proteins.
Transcription factors and gene regulatory
proteins have the capability of binding to DNA. This binding capability is based on the
interaction of amino acids of the protein with nucleotides of the DNA. The structure of
DNA-binding proteins falls into four categories as indicated below.
● Figure 7-2 Homeodomain Protein.
PIT-1 homeodomain
protein
PIT-1
gene 3p11.2
A. HOMEODOMAIN PROTEINS (Figure 7-2).
The homeodomain proteins consist of three
alpha helices (helix 1, 2, and 3) where helix 2
and 3 are arranged in a conspicuous helix-
turn-helix motif. The homeodomain proteins
contain a 60 amino acid long region within
helix 3 (called a homeodomain) that binds
specifically to DNA segments. The diagram
shows the three-dimensional structure of the
PIT-1 homeodomain protein (pituitary spe-
cific factor-1 or GHF-1) which is coded for by
the PIT-1 gene (also called POU1F1 gene) on
chromosome 3p11.2. The 60 amino acid
homeodomain of the PIT-l protein is coded for
by a 180 base pair sequence called the home-
obox sequence. PIT-1 protein binding at the
TI complex is required for transcription of the GH gene on chromosome 17q22, TSH
gene on chromosome 1p13, and the PRL gene on chromosome 6p22.2. A mutation in
the PIT-1 gene will result in the combined deficiency of growth hormone (GH), thy-
roid stimulating hormone (TSH), and PRL (prolactin) causing pituitary dwarfism.
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42
CHAPTER 7
B. LEUCINE ZIPPER PROTEINS (Figure 7-3).
The leucine zipper proteins consist of an alpha
helix that contains a region in which every sev-
enth amino acid is leucine which has the ef-
fect of lining up all the leucine residues on one
side of the alpha helix. The leucine residues al-
low for dimerization of two leucine zipper
proteins to occur and form a Y-shaped dimer.
Dimerization may occur between two of the
same proteins (homodimer; e.g., JUN-JUN) or
two different proteins (heterodimer; e.g., FOS-
JUN). The leucine zipper proteins contain a 20
amino acid long region that binds specifically
to DNA segments. The diagram shows the
three-dimensional structure of a leucine zip-
per protein (JUN) forming a leucine zipper ho-
modimer (JUN-JUN). L: leucine. Specific ex-
amples of leucine zipper proteins are
1.
C/EBP (CCAAT/enhancer binding pro-
tein)
which regulates the ALB gene on
chromosome 4q13.3 which encodes for al-
bumin and the SERPINA1 gene on chro-
mosome 14q32.1 which encodes for
1-
antitrypsin (or serpin peptidase inhibitor
clade A member 1).
● Figure 7-3 Leucine Zipper Protein.
L
COOH
Leucine zipper
protein (JUN)
+
DNA
binding
region
DNA
binding
region
NH
2
NH
2
Leucine zipper
protein (JUN)
Leucine zipper
Homodimer
(JUN-JUN)
COOH
L
L
L
L
L
L
L
L
COOH
NH
2
L
L
L
NH
2
COOH
L
L
L
L
2.
CREB (cyclic AMP response element binding protein)
which regulates the SST
gene on chromosome 3q28 which encodes for somatostatin and the PENK gene
on chromosome 8q23 which encodes for proenkephalin.
3.
FOS protein (Finkel osteogenic sarcoma virus)
which regulates various cell cy-
cle genes. The FOS protein is a product of the FOS gene (a proto-oncogene) on
chromosome 14q24.3.
4.
JUN protein
which regulates various cell cycle genes. The JUN protein is a prod-
uct of the JUN gene (a proto-oncogene) on chromosome 1p32.
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