by guest
`
`
`
`www.bloodjournal.orgFrom
`
`on March 28, 2016.
`
`For personal use only.
`
`B LOOD
`
`VOL
`
`73, NO 5
`
`REVIEW
`
`The
`
`J
`
`ournal
`
`of
`
`The
`
`American
`
`Society
`
`of Hematology
`
`APRIL
`
`1989
`
`A Review
`
`of
`
`the Molecular
`
`Genetics
`
`of
`
`the Human
`
`a-Globin
`
`Gene
`
`Cluster
`
`By D.R.
`
`Higgs,
`
`M.A.
`
`Vickers,
`
`A.O.M.
`
`Wilkie,
`
`I.-M.
`
`Pretorius,
`
`A.P.
`
`Jarman,
`
`and D.J. Weatherall
`
`D URING
`
`of
`for
`
`relatively
`THE
`been
`applied
`has
`biology
`synthesis
`and
`function,
`ture,
`Over
`ied most
`intensively.
`the
`of
`a detailed
`knowledge
`into
`insight
`and
`gained
`some
`stage
`tissue
`and
`developmental
`f3-globin
`nately
`regulated
`a-
`and
`Much
`of our
`understanding
`on
`come
`from
`observations
`occurring
`analysis
`of naturally
`and
`mia)
`/3-like
`(/3-thalassemia)
`summarizes
`our
`current
`function
`the
`normal
`lar
`basis
`a-thalassemia.
`
`in which molecular
`briefperiod
`disease,
`the
`struc-
`to human
`have
`been
`stud-
`of hemoglobin
`we have
`acquired
`past
`I 0 years
`the
`control
`of
`individual
`globin
`genes
`the mechanisms
`underlying
`the
`specific
`control
`of
`the
`coordi-
`gene
`clusters.
`expression
`of globin
`gene
`experimental
`systems
`and
`mutants
`ofa-like
`(a-thalasse-
`This
`globin
`chain
`synthesis.
`and
`knowledge
`of
`the
`structure
`a-globin
`gene
`cluster
`and
`the molecu-
`
`has
`the
`
`HISTORICAL
`
`PERSPECTIVE
`
`there
`
`fetalis
`
`syndrome
`
`1)t14
`
`the
`found
`
`in
`
`in
`in
`
`still
`carriers
`
`even
`proved
`a-thalassemia
`
`of
`
`to
`from
`
`a
`
`hydrops
`the Hb Bart’s
`2 and
`ca-thalassemia
`results
`from a-thalassemia
`I x a-thalassemia
`When
`in 1965
`directly
`it
`became
`possible
`to measure
`it was
`relative
`rates
`of a-
`and
`fl-globin
`chain
`synthesis
`synthesis
`that
`a progressive
`decrease
`in a-chain
`is
`Bart’s
`the Hb
`carriers
`a-thalassemia,
`Hb H disease,
`and
`of
`hydrops
`fetalis
`syndrome,
`respectively)5”8
`This
`is
`reflected
`in
`the
`relative
`levels
`of
`a-mRNA
`activity
`and
`quantity
`these
`syndromes.’92’
`However,
`using
`these
`techniques
`combination
`it
`often
`difficult
`distinguish
`unambiguously
`of
`non-thalas-
`semic
`individuals.
`further
`were
`a-thalasseniia
`genetics
`The
`molecular
`genes
`of
`over
`the
`number
`uncertainty
`complicated
`by
`22).
`reference
`(reviewed
`in
`individuals
`present
`normal
`in
`mutants
`structural
`of a-globin
`Based
`on observations
`it was
`loci2324
`but
`four
`a-chain
`clear
`that
`some
`individuals
`have
`could
`be
`polymorphism
`for
`other
`data
`suggested
`that
`there
`the
`number
`ofa
`genes
`even
`in apparently
`normal
`individuals
`25).
`was
`(reviewed
`in
`reference
`This
`important
`question
`fi-,
`were
`finally
`resolved
`when
`specific
`a-,
`and
`‘y-cDNAs
`normal
`isolated;
`solution
`hybridization
`studies
`showed
`that
`individuals
`have
`four
`a
`genes,
`and
`the
`a-thalassemia
`that
`syndromes
`usually
`resulted
`from
`the
`inheritance
`of 3, 2,
`0 a-globin
`genes.2629
`in
`a-thalassemia
`of
`genetics
`the
`These
`findings
`explained
`high
`findings
`of
`the
`explain
`Southeast
`Asia
`but
`did
`blood
`from
`cord
`judged
`as
`frequency
`of
`a-thalassemia,
`and
`the
`as Africa
`regions
`such
`surveys
`for Hb Bart’s
`in many
`I-lb H disease
`Middle
`East,
`where
`relatively
`uncommon
`is
`and
`the Hb Bart’s
`hydrops
`fetalis
`syndrome
`not
`occur
`does
`(reviewed
`in reference
`30).
`This
`ultimately
`explained
`by
`was
`understanding
`the
`structural
`organization
`the
`a-globin
`genes
`revealed
`by
`blot
`hybridization
`Normal
`
`not
`
`1 , or
`
`a
`
`of
`analysis.3’
`
`from a-thalasse-
`resulting
`syndromes
`clinical
`The major
`hydrops
`fetalis
`Hb
`Bart’s
`and
`the
`(Hb
`H disease
`mia
`and
`1950s
`early
`in the mid
`recognized
`syndrome)
`were
`first
`hemoglobins
`the
`abnormal
`of
`1960s
`through
`the
`association
`hypochromic
`microcytic
`anemia
`(Hb H and Hb Bart’s)
`with
`in
`the
`absence
`of
`iron
`deficiency.’4
`Identification
`tetra-
`of
`mers
`excess
`fl-like
`globin
`chains
`in these
`two
`syndromes
`of
`(Hb
`Bart’s
`-y4: Hb H,
`f34: and
`#{244}4tetramers)
`first
`provided
`evidence
`that
`these
`conditions
`result
`specifically
`from defects
`in the
`production
`ofa-globin
`chains59
`as proposed
`by
`Ingram
`and
`Stretton.’#{176}
`initially
`syndromes
`these
`of
`The
`genetics
`described
`hemoglobinopathies
`other
`in
`fusing
`than
`ofa-thalassemia
`the
`adult
`carriers
`because
`time.
`This was
`Bart’s.
`Hb
`H or Hb
`large
`amounts
`of
`either
`not
`produce
`individuals
`do not have
`relatives
`of
`affected
`the
`Although
`defined
`phenotype,
`a readily
`eventually
`shown
`that
`the
`it was
`of
`individuals
`with
`Hb H disease
`have
`raised
`levels
`offspring
`of Hb Bart’s
`(‘y(cid:1))
`in the
`neonatal
`period,t’
`and
`parents
`of
`the
`individuals
`with
`Hb
`H disease
`and
`the
`Hb
`hydrops
`Bart’s
`fetalis
`syndrome
`have
`mildly
`hypochromic,
`red
`microcytic
`cell
`indices’2;
`sometimes
`Hb H inclusions
`could
`be demon-
`strated
`in occasional
`red cells.’3
`Furthermore,
`such
`obligate
`carriers
`could
`be
`divided
`into
`mild
`(a-thalassemia
`2)
`and
`severe
`(a-thalassemia
`1)
`types
`depending
`on the
`of Hb
`Bart’s
`present
`at
`or
`the
`degree
`abnormality
`in
`the
`hematologic
`indices.
`1969
`it had
`shown
`Hb
`I-il
`disease
`from
`inheritance
`a-thalassemia
`x
`
`were
`
`more
`at
`
`con-
`that
`do
`
`the
`
`birth
`
`By
`the
`
`results
`
`of
`been
`of
`
`level
`
`that
`
`From
`
`the
`
`Department
`
`Molecular
`Medicine,
`
`Haematology
`John
`Radcliffe
`
`Unit.
`Hospital.
`
`Nuffield
`Head-
`
`29.
`
`1988;
`to DR.
`Nuffield
`Hospital.
`
`30.
`November
`accepted
`Higgs. MB.
`BS. M.R.C.
`Department
`ofClinical
`Headington,
`Oxford.
`
`1988.
`Molec-
`Medicine,
`England
`
`M.R.C.
`of Clinical
`England.
`Oxford.
`ington,
`September
`Submitted
`reprint
`requests
`Address
`ular
`Haematology
`Unit,
`Level
`7,
`John
`Radcliffe
`0X3
`9DU.
`1989
`
`0/
`
`by Grune
`
`& Stratton.
`
`Inc.
`
`I
`
`0006-4971/89/7305-0014$3.OO/O
`
`Blood. Vol 73. No 5 (April),1989: pp 1081-1
`
`104
`
`1081
`
`GENENTECH 2016
`GENZYME V. GENENTECH
`IPR2016-00383
`
`

`
`by guest
`
`
`
`www.bloodjournal.orgFrom
`
`
`
`For personal use only.on March 28, 2016.
`
`1082
`
`Table
`
`1 . Summary
`
`of Hematologic
`
`Findings
`
`in Individuals
`
`With
`
`a-Thalassemia
`
`Phenotype
`
`Equivalent
`of Functional
`
`Number
`a-Genes
`
`Level of
`at Birth
`Hb Bart’s
`1%)
`
`Normal
`
`a-Thaiassemiat
`trait
`(mild)
`
`a-Thaiassemiat
`
`(severe)
`trait
`HbHdisease
`
`Hb Bart’s
`Hydrops
`
`Fetalis
`
`4
`
`3
`
`2
`
`1
`
`0
`
`0
`
`0-2
`
`2-8
`
`10-40
`
`Hb H%
`(Inclusions)
`
`0
`
`(none)
`
`0
`
`(rare)
`0
`(occasional)
`1-40
`(many)
`
`present
`(present)
`
`MCV
`
`85-100
`
`75-85
`
`65-75
`
`60-70
`
`MCH
`
`-30
`
`(cid:1)(cid:1)26
`
`(cid:1)22
`
`.20
`
`1 10-120
`
`reduced
`
`HIGGS
`
`ET AL
`
`a/$ Globin Chain
`Ratiot
`Synthesis
`
`-.1.0
`
`09
`
`(cid:1)-0.4
`
`0.0
`
`These
`in Table 8.
`
`values
`
`vary
`
`con siderably
`
`depending
`
`on the
`
`age of
`
`the patient
`
`and
`
`the
`
`figures
`
`given
`
`are a guide
`
`to the
`
`indices
`
`seen
`
`in adul
`
`ts. More
`
`detail
`
`is given
`
`values
`tThese
`tThe mild
`
`and
`
`overlap
`
`to a considerable
`
`degree
`
`(see
`
`reference
`
`30
`
`and
`
`Table
`
`8).
`
`severe
`
`forms
`
`of a-thalassemia
`
`trait
`
`have
`
`often
`
`been
`
`referred
`
`to as ca-thalassemia
`
`2 and ce-thalassemia
`
`1 , respectively.
`
`have
`carriers
`two
`
`two
`for
`
`( - -/aa)
`
`(aa/
`a genes
`individuals
`(-a/
`three
`a-thalassaemia
`aa)
`and
`frequently
`Thus,
`a
`genes.
`aa)
`or
`Hb
`and
`of Hb
`H disease
`genotype
`encountered
`/ -
`only
`regions
`In some
`is
`fetalis
`Bart’s
`hydrops
`a-
`although
`therefore
`frequent,
`is
`-a
`determinant
`the
`( -
`Hb H
`neither
`a/
`a/aa
`and
`thalassemia
`is common
`occur.35’36
`syndrome
`hydrops
`fetalis
`disease
`the Bart’s
`was
`of a-thalassemia
`Thus
`I 980
`the molecular
`genetics
`analy-
`subsequent
`I ). However,
`understood
`in
`outline
`(Table
`defects
`molecular
`has
`that
`are many
`different
`there
`shown
`sis
`are
`now
`mutants
`this
`model.
`These
`simple
`underly
`that
`and
`a#{176}thalassemia
`to indicate
`chromosomes
`classified
`as
`is
`reduced
`(ak)
`absent
`(a#{176})output
`from
`in which
`there
`Furthermore,
`many
`non-thalassemic
`affected
`complex.
`serve
`are
`now
`known
`that
`not
`only
`ants
`the
`complex
`genetic
`analysis
`and
`anthropological
`useful
`markers
`for
`studies
`but
`also
`provide
`information
`on which
`segments
`the
`a-globin
`complex
`required
`its
`correct
`expression.
`
`on
`
`each
`
`-
`
`-
`
`-
`
`16
`
`chromosome
`have
`either
`the most
`
`is --/-a
`
`-
`
`a),
`
`-
`
`_(cid:1)“
`
`,(cid:1)
`
`a-Globin
`
`____________________
`Probes
`______
`
`Chromosome
`
`16
`
`on 16p13:-
`
`- a-Globin
`- PKD1
`- 0327
`- 090
`
`- 0129
`
`- 0133
`
`- 0136
`
`-P85
`
`_______
`
`\I
`
`on the
`shown
`are
`1 6p13.3(cid:1)”(cid:1)
`at
`genes
`a-globin
`The
`Fig 1 .
`region.
`from this
`probes
`1 6 specific
`of chromosome
`order
`The
`left.
`as described
`of
`the
`diagram.
`is shown
`on the
`right
`in reference
`46,
`used
`to provide
`-0129,
`-0327,
`0133
`CR1-090.
`-0136.
`The
`cosmids
`isolated
`from a chromosome
`1 6 only
`indicated,
`were
`the markers
`hybrid.
`P85 was
`isolated
`from a Los Alamos
`flow-
`mouse/human
`sorted
`chromosome
`1 6 library.
`PKD1
`denotes
`the
`disease
`locus
`for
`adult
`polycystic
`kidney
`disease.
`
`nor
`by
`
`a(cid:1)
`
`of
`
`or
`
`are
`
`for
`
`the
`van-
`as
`
`of
`
`LOCALIZATION
`
`OF THE HUMAN
`
`a-GLOBIN
`
`GENE CLUSTER
`
`a-
`
`the
`
`variants,
`fl-globin
`and
`with
`in a family
`studies
`Genetic
`are
`globins
`these
`that
`showed
`late
`1950s,
`in
`the
`performed
`by genetically
`encoded
`and
`fl-globin
`a-
`The
`loci.37
`distinct
`gene
`clusters
`were
`initially
`localized
`to chromosomes
`16 and
`I I ,
`of
`respectively,
`by observing
`the
`specific
`pattern
`cDNA-
`somatic
`DNA
`hybridization
`to
`human
`x mouse
`cell
`a-locus
`hybrids.38’39
`Subsequently,
`the
`localization
`of
`has
`of
`the
`been
`refined
`to
`the
`distal
`segment
`(p13.1-pter)
`short
`arm of chromosome
`I 6 by a variety
`of methods.(cid:1)#{176}(cid:1)43 Recently,
`two
`independent
`studies
`of
`individuals
`with
`unbalanced
`karyotypes
`have
`unequivocally
`located
`the
`a-locus
`to
`Giemsa
`negative
`band
`I6pl3.3
`the
`very
`tip of chromosome
`at
`l6,”(cid:1)(cid:1)
`although
`its orientation
`in the
`chromosome
`is not
`known
`(Fig
`The
`genetic
`localization.
`pter,
`including
`(PKD1),
`lie
`
`the
`
`yet
`
`1).
`
`map
`linkage
`Over
`3Ogenetic
`the
`locus
`centromenic
`
`for
`
`of
`
`to
`
`with
`consistent
`I 6p is also
`markers,
`localized
`to 16pl3.1
`adult
`polycystic
`kidney
`disease
`a-globin,
`none
`lies
`telomer-
`
`this
`1-
`
`(cid:1)
`

`
`by guest
`
`
`
`www.bloodjournal.orgFrom
`
`
`
`For personal use only.on March 28, 2016.
`
`THE
`
`HUMAN
`
`a-GLOBIN
`
`CLUSTER
`
`1083
`
`(cid:1)70kb
`
`5HVR
`
`0kb
`
`b inter-
`
`10kb
`
`20kb
`
`-
`
`Chromosome
`30kb
`
`22
`
`CHVR
`
`(cid:1)a2
`
`t(cid:1)ia1
`
`(cid:1) a2
`
`a!
`
`i(cid:1),p
`
`01
`
`3’HVR
`
`-0
`
`II
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`II
`
`I
`
`Alu-family
`
`repeats
`
`Embryo
`
`cy22’
`
`22
`
`1.
`
`I.
`
`i-etus
`a2’(cid:1)’2
`
`(cid:1)i(cid:1)i5ryo
`
`a2#{128}2
`
`(cid:1)AdUW
`
`Proteins
`
`synthesised
`
`Fig 2.
`represents
`lines.
`The
`indicated
`globin
`
`a-globin
`the
`of
`organization
`The
`CAP site.
`The
`the (cid:1)-globin
`mRNA
`positions
`of Alu family
`repeats
`are
`below
`the
`a complex.
`Each molecule
`chains.
`
`functional
`indicate
`boxes
`Filled
`complex.
`is shown
`on chromosome
`02 gene
`nonlinked
`complex.
`The
`hemoglobins
`below
`the
`shown
`of hemoglobin
`comprises
`a tetramer
`of
`two
`
`open
`and
`genes
`22. Hypervariable
`synthesized
`a-like
`(a-
`
`or
`
`Position
`pseudogenes.
`boxes
`by zig-zag
`are denoted
`regions
`at each
`stage
`of development
`are
`i’-) and two
`(fi-. ‘y-. h- or e-)
`a-like
`
`0
`
`THE
`
`STRUCTURAL
`
`ORGANIZATION
`
`OF
`
`THE
`
`a-GLOBIN
`
`CLUSTER
`
`in
`
`52)
`
`=
`
`51)
`the
`kb)
`G+C
`and
`
`the
`
`kb
`the
`are
`
`fractionation,49’(cid:1)#{176} pulsed
`and DNA
`sequence
`analy-
`suggests
`that
`26
`a-globin
`genes
`and
`flanking
`the
`cluster
`rich
`DNA
`segment
`surrounding
`DNA
`the
`with
`associated
`such
`early
`replicating
`(re-
`a Giemsa
`negative
`associated
`with
`non-
`contain
`many
`Alu-
`of
`genome
`are
`the
`of
`“housekeeping
`thus
`constitute
`to
`in
`contrast
`normal
`nucleotide
`in
`reference
`54]),
`late
`in nonhe-
`
`an
`the
`
`and
`
`a genes
`pseudogenes
`
`(a2
`
`of
`
`the
`
`0 gene
`
`copy
`processed
`a truncated,
`and
`of a
`stream
`22.(cid:1)#{176}
`chromosome
`found
`on
`was
`(02)
`family
`repeated
`tandemly
`cluster
`contain
`the
`of
`regions
`Several
`identified
`were
`first
`(minisatellites).
`They
`of DNA
`segments
`the
`at
`3’ end
`of
`the
`regions
`(HVRs)
`located
`as hypervariable
`(cid:1)2
`a,1’(cid:1)l genes
`and
`3’HVR),
`between
`complex
`(a-globin
`(IVS
`1 and
`IVS
`and within
`the
`introns
`(interzeta-HVR)
`(summarized
`in Fig
`((cid:1)‘-intron
`HVRs),6t’62
`genes
`ofthe
`c-like
`have
`recently
`identified
`a
`further
`hyper-
`2). We
`and
`Table
`(cid:1)2
`region
`approximately
`kb
`upstream
`of
`(called
`variable
`70
`the
`a-globin
`5’HVR.63
`The
`structure
`of
`these
`regions
`and
`2.
`is of
`interest
`their
`interrelationships
`are
`shown
`in Table
`that
`such
`regions
`be
`particularly
`frequent
`at
`the
`telo-
`may
`meres
`of human
`chromosomes.
`
`2)
`2
`
`the
`
`It
`
`EVOLUTION
`
`OF THE a-GLOBIN
`
`FAMILY
`
`started
`million
`evolved
`a variety
`by
`conversion,
`cluster
`
`a
`
`density
`from buoyant
`Evidence
`field
`gel
`electrophoresis
`studies,5’
`sis
`(summarized
`reference
`segment
`of DNA
`containing
`extended
`cloned
`segment
`(1 50
`part
`of
`a very
`long
`(>200
`kb)
`(isochore).
`The
`a-globin
`cluster
`have
`many
`of
`the
`characteristics
`regions;
`they
`are G+C
`rich
`(6O%),(cid:1)’
`viewed
`in
`reference
`present
`within
`band”’45
`(see
`previous
`section),
`are
`methylated
`CpG-rich
`islands53’54
`and
`family
`repeats.55
`In
`general
`such
`regions
`thought
`to
`contain
`a
`high
`proportion
`genes”;
`the
`tissue
`specific
`a-globin
`genes
`interesting
`exception.
`These
`findings
`are
`human
`/3-globin
`cluster,
`which
`has
`a more
`composition
`(G+C
`[reviewed
`39.5%
`contains
`no CpG-rich
`islands,
`and
`replicates
`52).
`matopoietic
`cells
`(reviewed
`in reference
`The
`a-globin
`cluster
`includes
`the
`duplicated
`al),
`an embryonic
`a-like
`gene
`((cid:1)2),
`three
`
`(XI,
`arranged
`al
`-01
`family
`
`4’a2,
`
`-
`
`of
`
`a/’al),
`in
`the
`3’
`(Fig
`small
`
`and
`order
`
`2).56.58
`cytoplasmic
`
`a gene
`5’
`Recently,
`RNAs
`
`of
`
`-
`
`(cid:1)2
`
`-
`
`undetermined
`(cid:1)1(cid:1)l
`-
`a pseudo
`was
`
`(cid:1)a2
`
`function
`i/’aI
`for
`
`-
`
`gene
`identified
`
`(01)
`a2
`
`-
`
`-
`
`Ro
`the
`down-
`
`The
`from
`ago.67
`series
`processes
`position).
`species
`mals
`and
`(Fig
`MY
`ago,58
`
`genes
`f3-globin
`and
`a-
`ancestral
`approximately
`each
`other
`500
`subsequently
`family
`has
`Each
`gene
`modified
`of
`gene
`duplications
`deletion,
`gene
`(divergence,
`of
`the
`a-globin
`Comparisons
`ancestral
`a-globin
`the
`suggest
`that
`included
`a c-gene,
`(85 MY
`ago)
`(cid:1)‘-5’a-3’a-0).(cid:1)
`a 0 gene
`(proto
`divergence
`3)
`that
`the
`(cid:1)/a
`0/a
`divergence
`ago62
`and
`the
`although,
`because
`01 may
`
`diverge
`to
`(MY)
`years
`through
`of
`genetic
`and
`retro-
`from
`several
`family
`in mam-
`duplicated
`a-genes,
`It has
`been
`calculated
`place
`approximately
`400
`occurred
`260
`to 280 MY
`not
`serve
`as
`a globin
`gene
`
`took
`
`Table
`
`2.
`
`Summary
`
`of
`
`the Repeat
`
`Elements
`
`Within
`
`the
`
`a-Globin
`
`Hypervariable
`
`Regio ns (HVRs)
`
`Sequence
`
`Unit Size
`tbp)
`
`Number
`Repeats
`
`of
`Range)
`
`Enzymes
`Used
`
`Commonly
`to Detect
`Alleles
`
`Reference
`
`64
`
`63
`
`62
`
`Rsat. Dvhl
`
`Sect, Pu-till
`
`Sect. Seu96i
`
`6 1 . 65.
`
`66
`
`Reef.
`Pvuit.
`Sect. Pvuti
`
`Hinti
`
`6 1 . 64
`62
`
`Name
`
`Core
`
`5’HVR
`
`tHVR
`
`tivisit
`
`IZ-HVR
`
`3’HVR
`
`tHVR ttVIS2t
`
`GGGGAGCATTCAGGAGGCCTtCCCGGAGGTAGGGTGGTGGGAAGAA000G
`
`GNGGGGNACAG
`I I I I
`I III
`I
`- TCAGCGT
`II
`I I (cid:1) I
`I (cid:1)
`RGGAGGGG-ACAGTG
`I I (cid:1) I
`I I I I
`TGTGAGGGTGCccGGGACGGCT1GTGGGGCACAG-GT
`I I I I
`I
`I
`II I
`ACACGGGGGG.AACAGCG
`CGGGG
`
`I
`
`57
`
`14
`
`36
`
`1 7
`5
`
`I
`
`5-55
`
`12-30
`
`1 2-65
`
`70-450
`35-52
`
`Each hypervariable
`
`region
`
`comprises
`
`a segment
`
`of DNA containing
`
`a variable
`
`number
`
`of
`
`repeats
`
`of unit
`
`sizes ranging from 5 to 57 bp as indicated,
`
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`

`
`by guest
`
`
`
`www.bloodjournal.orgFrom
`
`
`
`For personal use only.on March 28, 2016.
`
`HIGGS
`
`ET AL
`
`proto
`a gene
`divergence
`The
`4’al
`al
`and
`a2
`ago73
`after
`but
`before
`
`3’ a genes,
`duplica-
`from
`(proto
`genes
`the
`the
`
`has
`
`derived
`are
`a2/al
`proto
`
`1084
`
`S T
`
`otes
`
`0
`
`i2
`
`2
`
`10
`
`17
`
`16
`
`OENES
`
`COMPARED
`
`SUBSTITUTION
`
`S
`
`PER
`
`SITE
`
`S
`
`MIS
`
`MATCH
`
`0
`
`F
`
`AMINO
`
`ACID
`
`Replacesent
`
`Silent
`
`E(cid:1)onl
`
`Exon2
`
`(cid:1)(cid:1)p3
`
`a2
`
`vs
`
`a2
`
`vs
`
`(cid:1)2
`
`os
`
`01
`
`vs
`
`al
`
`(cid:1)2
`
`d#(cid:1)l
`
`a2
`
`81
`
`vs
`
`(cid:1)2
`
`0.00
`
`0.38
`
`0.01
`
`0.28
`
`0.55
`
`0.22
`
`0.00
`
`0.60
`
`0.00
`
`0.67
`
`0.72
`
`o.1i8
`
`0
`
`66
`
`6
`
`55
`
`71
`
`38
`
`0
`
`37
`
`0
`
`31i
`
`41
`
`47
`
`0
`
`33
`
`2
`
`35
`
`40
`
`50
`
`for
`
`yet been
`not
`between
`this
`a gene
`(Fig
`3)
`a
`considerable
`radiation.
`and
`al
`identical
`that
`
`a2
`
`genes
`each
`have
`of
`
`5’ a and
`proto
`from the
`mals
`that
`from a further
`results
`pair
`the
`human
`occured
`after
`3’ a gene
`that
`tion
`of
`the
`72 MY
`ago).
`lineage
`to
`the
`prosimian
`(52
`the
`functional
`from
`to diverge
`started
`S’a)
`60 MY
`inactivated
`45 MY
`and was
`ago
`and
`higher
`primates,
`of prosimians
`divergence
`and
`great
`apes.
`of monkeys
`divergence
`gene
`history
`of
`the
`4ia2
`The
`evolutionary
`the
`extensive
`differences
`elucidated.
`However,
`and
`the
`functional
`nonfunctional
`pseudogene
`been
`diverging
`suggest
`they
`have
`that
`time,57
`since
`before
`the mammalian
`possibly
`DNA
`analysis
`of
`the
`human
`sequence
`shows
`have
`remained
`virtually
`that
`they
`fact
`other
`during
`evolution74’75
`despite
`the
`both
`a-globin
`diverged
`as
`expected
`from
`the
`other
`species.
`Sequence
`homology
`between
`gene
`has been
`similarly
`maintained.62
`This
`in-tandem
`is
`called
`concerted
`evolution,
`through
`related
`genetic
`mechanisms,
`two
`and
`gene
`conversion
`(reviewed
`with
`respect
`reference
`76).
`The
`intermediates
`of
`crossover
`[ -a,
`or
`triplicated
`mosomes
`with
`single
`are
`genes)
`frequently
`observed
`in extant
`human
`below).
`(see
`of
`The
`structure
`during
`further
`modified
`copies
`position
`of many
`2). The
`elements55’72’75
`(Fig
`within
`segments
`of DNA
`is interesting
`clear.
`However,
`it
`in the
`(cid:1)1ntron
`14 bp repeat
`present
`suggesting
`intron
`of
`the
`goat”
`have
`preceeded
`the mammalian
`
`to
`they
`sequence
`4’(cid:1)I
`(cid:1)2
`and
`of evolution-
`which
`occurs
`crossover
`fixation
`to the
`a-genes
`fixation
`(chro-
`(cid:1)]
`[aaa,
`populations
`
`the
`type
`
`in
`
`-fl
`
`the
`
`(cid:1).
`
`human
`evolution
`of
`
`the
`origin
`the
`
`cluster
`insertion
`family
`of
`tandemly
`regions
`similar
`present
`structures
`
`been
`has
`by retro-
`repetitive
`repeated
`is
`not
`to the
`in the
`may
`
`a-globin
`through
`Alu
`the
`of
`minisatellite
`that
`a sequence
`HVR
`is also
`that
`such
`radiation.
`
`THE
`
`STRUCTURE
`
`AND
`
`EXPRESSION
`
`OF
`
`INDIVIDUAL
`
`a-LIKE
`
`GLOBIN
`
`GENES
`
`are
`
`58%
`and
`
`a
`a
`
`like
`
`and
`
`of
`a series
`through
`evolved
`has
`family
`gene
`a-globin
`The
`now,
`that
`such
`divergence
`sequence
`and
`duplications
`gene
`(Fig
`structure
`in polypeptide
`similarities
`although
`there
`in
`homology
`show
`only
`and
`(cid:1) genes
`a
`3),
`the
`functional
`are
`a2
`genes
`al
`the
`acids.
`In
`contrast
`their
`141
`amino
`and
`proteins,
`identical
`(Fig
`3),
`encode
`highly
`homologous
`(17%
`regions
`3’ noncoding
`IVS2
`and
`their
`only
`differ
`within
`share
`genes
`the
`a-like
`all
`Curiously,
`of
`divergence).74
`with
`which
`is associated
`G + C content,
`high
`surprisingly
`extremes
`in base
`composition
`usage.78
`Similar
`bias
`in codon
`noted
`for
`other
`genes
`that,
`and
`codon
`usage
`have
`been
`GC-rich
`isochores.49
`the
`a-gene
`family,
`lie within
`between
`Comparisons
`of many
`globin
`genes
`within
`have
`been
`species
`demonstrate
`that
`some
`structural
`features
`( I to
`conserved.79’80
`In general,
`the
`globin
`genes
`are
`compact
`2 kb, Table
`3)
`and
`are
`divided
`into
`three
`exons
`(protein
`coding
`regions)
`by
`two
`noncoding
`intervening
`sequences
`are
`of
`or
`(IVS
`introns).
`The
`positions
`the
`introns
`(Fig
`4)
`conserved
`in all globin
`genes
`and may
`reflect
`the
`early
`(>500
`MY
`ago)
`evolutionary
`events
`that
`brought
`together
`the
`functional
`domains
`the
`protein.8’
`For
`example,
`2
`
`of
`
`exon
`
`6a1
`
`vs
`
`a2
`
`6c2
`
`vs
`
`a2
`
`(cid:1)se2
`
`vs
`
`t2
`
`0.511
`
`0.61
`
`0.66
`
`0.9(cid:1)i
`
`69
`
`91
`
`60
`
`57
`
`(cid:1)8
`
`48
`
`58
`
`62
`
`I
`
`a2,a
`
`1
`
`1(cid:1) -like
`
`genes
`
`(cid:1)-6O
`‘100
`
`500
`
`3.
`
`Fig
`(above).
`in
`acids
`described.
`references
`ary
`events
`for
`these
`73.
`
`a-like
`
`genes
`the
`of
`regions
`coding
`the
`of
`Comparison
`amino
`of
`mismatch
`percent
`and
`site
`per
`Substitutions
`previously
`calculated
`as
`were
`regions
`the
`coding
`are
`table
`derived
`from
`this
`of
`for most
`The
`data
`of
`timing
`the evolution-
`57 and 58. Below,
`the proposed
`summarized.
`The
`dates
`in the
`a-globin
`gene
`family
`are
`divergence
`times
`are
`from references
`57. 58. 62,
`67. and
`
`to fulfil
`
`another
`
`function,
`
`the
`
`rapidly
`evolved
`have
`may
`and
`be an
`overestimate.69
`may
`latter
`are
`three
`further
`duplications
`least
`At
`family.
`present
`day
`human
`a-gene
`the
`for
`the
`that
`cluster
`in
`apes
`shows
`a-globin
`and
`the
`divergence
`of
`human
`predates
`based
`MY
`ago.7#{176}Provisional
`be
`mated
`.(cid:1).5
`regions
`the
`horse
`and
`human
`(cid:1)-globin
`of
`comparisons
`gests
`that
`have
`even
`predated
`the
`separation
`it may
`lineages.7’
`However,
`more
`precise
`primate
`equid
`and
`of
`the
`duplication
`event
`cannot
`the
`timing
`mates
`of
`insufficient
`data
`from
`other
`species
`there
`are
`made
`since
`the
`human
`(cid:1)2
`and
`a(cid:1)l
`genes
`(Fig
`direct
`comparison
`of
`that
`they
`have
`recently
`undergone
`reference
`62)
`shows
`(see
`below)
`has
`eradicated
`gene
`conversion
`event
`that
`DNA
`sequence
`differences
`by which
`to calculate
`the
`of
`the
`duplication.
`The
`t(cid:1)’a I -a2-a
`genes.
`Sequence
`humans
`and
`galago
`a(cid:1)f’al
`is derived
`from
`gene.72
`Thus,
`unlike
`
`to
`
`to account
`required
`of
`the
`Mapping
`c-gene
`duplication
`chimpanzee
`(esti-
`data
`on
`sug-
`the
`esti-
`be
`and
`3 and
`
`yet
`
`of
`
`a
`
`any
`timing
`
`in
`
`S’a-3’a
`proto
`a-genes
`pseudogene
`3’a
`from
`proto
`of many
`mam-
`
`is derived
`of
`
`1 cluster
`comparisons
`the
`show
`that
`the
`proto
`S’a
`the
`duplicated
`
`the
`from
`orthologous
`nonfunctional
`gene
`and
`al
`a genes
`
`

`
`by guest
`
`
`
`www.bloodjournal.orgFrom
`
`
`
`For personal use only.on March 28, 2016.
`
`THE HUMAN a-GLOBIN
`
`CLUSTER
`
`1085
`
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`www.bloodjournal.orgFrom
`
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`For personal use only.on March 28, 2016.
`
`1086
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`HIGGS
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`3)
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`
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`
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`3). The
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`suggested
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`01
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`(t/’02)
`been
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`of mRNA
`transcripts
`have
`transcribed
`and
`(see
`later).
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`it
`is not
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`clear
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`“ATA”
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`of0l
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`promot-
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`ofOl
`mRNA
`not
`been
`assessed,
`and
`this may
`has
`be
`particularly
`important
`view
`of
`the
`divergence
`of
`the
`in
`extended
`motif
`the
`consensus
`(Table
`3).
`from
`Although
`sequence
`comparisons
`suggest
`that
`01
`
`ATG
`DNA
`
`the
`
`region
`
`of
`
`600
`
`bp
`
`beyond
`
`the
`
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`
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`
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`genes
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`Tran-
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`
`in
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`a
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`
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`
`through
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`
`and
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`the
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`downstream
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`
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`
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`
`to
`
`a
`
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`
`30
`
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`
`downstream
`
`from
`
`sequence.e*(cid:1)J*
`
`The
`
`“CAAT”
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`which
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`
`in
`
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`
`bases
`
`the
`
`level
`
`of
`
`transcription.
`
`is
`
`located
`
`approximately
`
`80
`
`bp
`
`upstream
`
`of
`
`the
`
`CAP
`
`site.e7(cid:1)
`
`A second
`
`type
`
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`
`found
`
`upstream
`
`of
`
`the
`
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`genes
`
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`
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`
`similar
`
`to
`
`those
`
`found
`
`associated
`
`with
`
`the
`
`promoter
`
`(Table
`
`regions
`
`3)
`
`of
`
`which
`
`bind
`
`the
`
`transcription
`
`factor
`
`SP1
`
`a
`
`housekeeping
`
`genes.
`
`Following
`
`transcription
`
`of
`
`the
`
`globin
`
`genes.
`
`modifications
`
`take
`
`place
`
`CAP
`
`at
`
`both
`
`ends
`
`of
`
`the
`
`pre
`
`mRNA.
`
`The
`
`5’
`
`end
`
`is modified
`
`by
`
`the
`
`structure
`
`(m7G).
`
`The
`
`3’
`
`ends
`
`of
`
`mRNAs
`
`are
`
`formed
`
`by
`
`endonucleolytic
`
`cleavage
`
`of
`
`the
`
`pre
`
`mRNA.
`
`Following
`
`cleavage.
`
`poly(A)
`
`tracts
`
`of
`
`approximately
`
`200
`
`nucleotides
`
`[(A)n]
`
`are
`
`synthe-
`
`adenosine
`
`residues
`
`on
`
`the
`
`3’
`
`end
`
`of
`
`the
`
`of
`
`First,
`stages.
`in two
`proceeds
`splicing
`that
`shown
`have
`vitro
`5’ phosphoryl-
`and
`the
`site
`occurs
`(1GU)
`the
`5’ splice
`at
`cleavage
`of an A residue
`2’OH
`the
`intron
`is joined
`to the
`of
`ated
`terminus
`3’ splice
`site.
`In the
`located
`20 to 40 nucleotides
`upstream
`of
`the
`18
`(IVS
`II) nucleotides
`genes
`these
`A residues
`lie 19
`(IVS
`I) and
`generates
`a linear
`RNA
`upstream
`the
`splice
`site.(cid:1)
`This
`step
`the
`precursor
`and
`the
`comprising
`the
`upstream
`segment
`of
`consisting
`of
`the
`intron
`so-called
`lariat
`intermediate.
`a structure
`The
`two
`intermediates
`of
`and the downstream
`part
`the
`pre RNA.
`are
`retained
`in a nucleoprotein
`complex
`referred
`to as the
`spliceo-
`some.
`Cleavage
`then
`takes
`place
`at
`the
`3’ splice
`site
`(AG1)
`and the
`3’
`end
`of
`the
`upstream
`exon
`is
`joined
`to
`the
`5’
`end
`of
`the
`downstream
`exon.
`Finally.
`the
`intron
`is released
`as a lariat.
`which
`is rapidly
`degraded
`in vivo. Nuclear
`mRNA
`is transported
`through
`the nuclear
`membrane
`to the
`cytoplasm
`where
`it
`is translated
`into
`protein.
`The
`AUG
`codon
`that
`lies
`nearest
`to the
`5’ end
`the
`of
`message
`(I)
`is the
`functional
`initiator
`codon.
`Sequences
`flanking
`AUG
`(CCRCCAUGG)
`have
`also
`been
`shown
`to
`influence
`the
`(T)
`efficiency
`of
`translation.(cid:1)’M
`Termination
`of
`translation
`is
`effected
`by nonsense
`codons.
`UAA.
`or UGA.
`In the box below.
`the
`important
`structural/functional
`residues
`of
`the
`a-like
`genes within
`the
`three
`exons
`(shaded)
`indicated
`by vertical
`bars
`(modified
`with
`permissionN).
`
`are
`
`downstream
`
`(reviewed
`
`in
`
`reference
`
`90).
`
`During
`
`the
`
`noncoding
`
`introns
`
`are
`
`removed
`
`and
`
`the
`
`the
`
`exons
`
`are
`
`splicing
`
`joined
`
`protein
`
`to
`
`produce
`
`an
`
`uninterrupted
`
`mRNA
`
`that
`
`encodes
`
`the
`
`entire
`
`(reviewed
`
`in
`
`reference
`
`91
`
`).
`
`The
`
`introns
`
`are
`
`bounded
`
`consensus
`
`sequences
`
`(denoted
`
`by
`
`S).
`
`the
`
`(cid:1)GU
`
`of
`
`the
`
`5’ consensus
`
`and
`
`AGI
`
`of
`
`the
`
`3’
`
`consensus
`
`being
`
`highly
`
`conserved.
`
`Studies
`
`by
`
`in
`
`sized
`
`RNA
`
`by
`
`the
`
`addition
`
`of
`
`at
`
`the
`
`site
`
`of
`
`cleavage.
`
`RNA
`
`from
`
`beyond
`
`the
`
`cleavage
`
`rapidly
`
`degrades.
`
`At
`
`least
`
`three
`
`sequences
`
`may
`
`be
`
`involved
`
`point
`
`in this
`
`process;
`
`the
`
`motif
`
`AAUAAA
`
`(denoted
`
`by
`
`A)
`
`is
`
`found
`
`10
`
`to
`
`30
`
`nucleotides
`
`downstream
`
`of
`
`the
`
`site
`
`of
`
`poly(A)
`
`addition
`
`(usually
`
`CA)
`
`often
`
`followed
`
`by
`
`a
`
`‘G/T
`
`clusterS’
`
`10
`
`to
`
`30
`
`nucleotides
`
`process
`
`of
`
`(cid:1)
`(cid:1)
`(cid:1)
`

`
`by guest
`
`
`
`www.bloodjournal.orgFrom
`
`
`
`For personal use only.on March 28, 2016.
`
`THE HUMAN a-GLOBIN
`
`CLUSTER
`
`1087
`
`strong
`
`it
`
`is
`
`has
`
`evolved
`that
`the
`would
`not
`gene.
`
`patterns
`nuclear
`
`of methylation,53’54’(cid:1)2
`attachmenttt3
`between
`
`and
`the
`
`a-
`
`marked
`and
`
`differences
`fl-globin
`genes.
`
`in
`
`EXPRESSION
`DURING
`
`a-LIKE
`THE
`OF
`DEVELOPMENT
`
`GENES
`
`the
`
`of
`
`end
`during
`(Hb
`Hb
`to fetal
`(Hb
`A2,
`both
`in
`line
`
`a-
`
`and
`there
`
`fl-loci
`is
`
`(up
`
`in
`
`(from
`assays,
`Furthermore,
`fetal
`
`in
`
`of
`
`toward
`the
`of
`activation
`Following
`transition
`a
`gestationt14
`week
`of
`third
`the
`embryonic
`of
`the
`production
`from
`development
`Hb Gower
`2, a2e2)
`Hb Gower
`1,
`Portland,
`(cid:1)2(cid:1)2;
`(cid:1)2’Y2;
`F a2y2)
`Hb (Hb
`(Hb A,
`and minor
`to the major
`a(cid:1)fl2)
`(Fig
`Whereas
`adult
`hemoglobins
`a2b2)
`2).79809798t(cid:1)(cid:1)
`in primitive
`a-
`and
`(cid:1)-globins
`are
`synthesized
`erythroblasts
`of gestation),
`the
`yolk
`sac
`to 6 to 7 weeks
`definitive
`erythroblasts
`the
`liver
`almost
`exclusively
`synthesize
`more
`a-globin
`6 weeks
`onward).”5
`Using
`sensitive
`mRNA
`0 transcripts
`are
`detected
`in yolk
`sac
`and
`fetal
`liverY(cid:1)
`small
`amounts
`of
`(cid:1)-mRNA
`are
`present
`throughout
`life,82t16
`and
`(cid:1)-globin
`(as Hb
`Portland)
`has
`been
`detected
`the
`cord
`bloods
`nonthalassemic
`new-
`born.’8
`Although
`identical,”7
`in
`their
`amounts
`cytes.”8”9
`mRNA
`throughout
`at
`the
`level
`the
`ribosome
`they
`have
`should
`be
`dominant
`a-globin
`has
`enabled
`the
`a2
`and
`cally
`shows
`protein
`than
`(3’)
`a-locus
`findings
`type
`a2
`
`of
`
`the
`the
`
`al
`
`are
`
`a2
`3:1
`
`of
`that
`
`a
`of
`
`-k
`
`3).
`
`6
`a
`
`the
`the
`by
`down-
`
`the
`
`of
`
`3.
`
`constraints58
`selective
`under
`gene
`acid
`polypeptide
`amino
`141
`predicted
`thought
`hemoglobin.69
`a functional
`encode
`sequence
`t/i(cid:1)1
`The
`for
`unit
`extends
`duplication
`The
`(cid:1)2-(cid:1)t’(cid:1)1
`repeat
`for
`some
`except
`and,
`2,000
`bp
`approximately
`there
`are
`two
`genes
`of
`the
`introns
`in the
`variation
`sequence
`of which
`region,
`one
`this
`within
`changes
`base
`pair
`only
`six
`at codon
`TAG)
`(CAG
`mutation
`a nonsense
`introduces
`evidence
`this
`On
`t(cid:1)(cid:1)#{149}1gene.62
`of
`the
`is
`described
`as
`it
`direct
`globin
`synthe-
`it clearly
`cannot
`since
`gene
`pseudozeta
`canonical
`sequences
`it
`contains
`of
`the
`all
`though
`sis
`even
`Surprisingly,
`how-
`expression
`(Table
`for
`gene
`required
`is
`restored
`the
`the
`nonsense
`mutation
`to
`when
`even
`ever,
`(GAG)
`by
`a
`gene
`conversion
`event,
`stable
`codon
`normal
`transcripts
`from this
`gene
`(referred
`to as
`rather
`(cid:1)l
`mRNA
`are
`still
`not
`detected,
`suggesting
`that
`sequences
`s(cid:1)1)
`than
`outside
`of
`the
`conversion
`unit
`influence
`lying
`may
`be
`expression
`this
`gene.82
`This
`may
`explained
`of
`presence
`a negative
`regulatory
`element
`to
`5’
`stream
`t,t’(cid:1)1)
`but
`not
`the
`upstream
`((cid:1)2)
`locus.t02
`or
`((cid:1)I
`by
`The
`(cid:1)C’a1 gene
`is inactivated
`The
`and
`i,t’a2 genes.
`i,&al
`in
`a variety
`transcriptional
`control
`mutations
`as
`indicated
`of
`there
`the
`Table
`In addition
`is a 23 bp deletion
`between
`boxes
`(1 to
`“CAAT”
`and
`“ATA”
`and
`several
`small
`deletions
`mRNA
`the
`20
`bp)
`that
`alter
`reading
`frame
`introducing
`codons
`the
`terminate
`that
`would
`translation.73
`Although
`in a cell
`promoter
`region
`is capable
`of directing
`transcription
`the mutant
`free
`assay
`and
`in
`transient
`expression
`systems,
`no stable
`poly(A)
`addition
`site
`is apparently
`nonfunctional;
`cells.’#{176}3The
`are
`t’al
`transcripts
`detected
`in human
`erythroid
`and
`is clearly
`gene
`completely
`lacks
`a promoter
`region
`ti’a2
`inactivated
`by
`frameshift
`mutations
`a variety
`of muta-
`plus
`tions
`in the
`transcriptional
`control
`sequences.57
`
`COORDINATED
`
`EXPRESSION
`
`OF
`
`THE
`
`a-
`
`AND
`
`fl-LIKE
`
`GLOBIN
`
`GENES
`
`the
`
`a-
`
`the
`
`is
`
`is
`
`a
`
`Once
`during
`activated
`been
`have
`loci
`fl-globin
`and
`there
`to suggest
`that
`evidence
`is
`little
`there
`development,
`nota-
`subsequent
`regulation.’#{176}(cid:1)”#{176}5Most
`in their
`any
`interplay
`excess
`type
`f3-thalassemia,
`globin
`chains
`of one
`bly
`in a-
`and
`in large
`synthesized
`amounts
`despite
`a deficit
`continue
`to be
`in reference
`of
`the
`other
`(reviewed
`104).
`In this
`context,
`it
`lines
`of
`interesting
`that
`several
`evidence
`tentatively
`suggest
`that
`the
`processes
`underlying
`a-
`and
`fl-globin
`expression
`are
`somewhat
`different.
`For
`example,
`in
`transient
`expression
`systems
`a
`globin
`genes
`are
`expressed
`in
`enhancer
`an
`fi gene
`of
`independent
`manner,
`whereas
`expression
`the
`only
`occurs
`in the
`presence
`of
`the
`SV4O
`enhancer.’00”#{176}7 In
`stably
`transformed
`mouse
`erythroleukemia
`(MEL)
`cell
`lines
`relatively
`globin
`appears
`to be expressed
`at
`high
`levels
`before
`fl-globin
`and
`after
`induction,t08
`whereas
`the
`gene
`is expressed
`to
`more
`appropriately
`with
`respect
`although
`0910
`both
`genes
`are
`expr