CELLULAR DIFFERENTIATION

IN HIGHER EUKARYOTES

During the development of a higher eukarvotea

Single cellethe zvgotegiveś rise by mitotic cell divi

sions to à vast

nerve cells, bone cels, blood cells, etc.) with highly

array of cell svpes (in animalsş skin cells

cells, etc.) with nigil

divergent morpho

ular compositions. These different cell types are en

nrghty speciaized, carrying out only fea specific

metabolic functuons. For example, red bloed cells are

highiy specialized f

hemoglobin. In amphibians.ror

example, nuclei from differentiated ces. awhich the

planted into enucleated eggs (eggs from which the

original nuclei have been removed) and shown ta

ne

of the geneuc information required for the normal

during the

aibian nuclei-donor cells. During the development of a complex plarit o

ene expression has been shown to

lar

ated in different

instances at essentially all possible

processing mRNA

leve's-ranscription, pre-mRNA

transport, MRNA stability, translation, posttranslational

protein processing, protein stability and enzyme funçtion.

Many of the developmental processes in higher

eukaryotes seem to be controlled, at least in part, by

preprogrammed circuits of gene ex

cases, some event (such as release of a hormone in the

bloodstream or fertilization of an egg) triggers the

n these

expression of one set of genes The product (or prod

ucts) of one (or more) of these genes funcions Dy

turning off the transcription of the first set of genes

and/or turning on the transcripion of a second set of

genes. In urn, one or more of the products of the

second set acts by turning on a third set, and so on. In

these cases, the sequential expression of genes is

genetically preprogrammed, and the genes cannot

usually be turned on out of sequence.

In eukaryotes we knory that homones can trigger

the sequential expression of sets of genes. i. addition,

we Know that regulatory génes are nvoves in th

control of patterns of difierentiation. In some cases, we

know nar regutatory erements called enbancers and

silencers modulate levels of gene expression from

nearby promoters.

Belum lengkap,,, resulted from the application of recombinant DNA an

gene cloning technologi

overlook some of the elegant neoclassical studies

use us t

entally regulated gene expression. The strik

ing pictures of transcription on lampbrush chromo

somes and of the amplification cof nlbosemal R

genes in ampfibian oocytes are

ples

that predated the recombinant DNA epoch of biology.

Transcription on Lampbrish Chromosomes

in Amphi

In all higher organisms studied so far, the fertilizatior

in protein synthesis, followed by the rapid nudear an

of a mature egg by a sperm triggers a dramatis increan

cell divisions'of early cleavage stages of embryogene

sis. In most eukaryotes, this prote

esis is n

ents

uired f

or protein Smthesis du

zation. Géne

pre-mRNA molecules, must theeforeve stored

eg in a dtn ant state.Translaign of hese preforme

mRIVA molecules mus

erents associared with fea

Therefore, the informa.ional nolecules that dire

states fol

fergilization imust be synthesiuei diring gen

of oögenesis in yertebrates, párticularb

protein synthesis curing the early cleavage

amphibians,revel hat extensi

during prophase I (Specificaly diploteue) of me

scrip

ion occur

During this stage, the chror.csomes exist as arg

lampbrush structures,

rRNA Gene Amplification

Despite the rapid initktion of protein synthesis follow

ing fertilization, no rRRA is synthesized 讥 amphibian

embryos until the gastrata stage Thus means har large

amounts of rRNA mt also be synthesized during

vast quantities of ribosomes, on tihe order of 101 per

enormous amounts of a particular gene transcript (the

resulted in the evolution of a novel mechanism of

the rRNA genes are seloatively amplified about a thou

is. in fact
gs of amphibians c

maure egs. The

for the synthesis of such

40S amphibian rRNA precursor) in a single cell

specific gene amplification in amphibian

sandfold to facilitate thesynthes

ties of rRNA stored in saure eges

in Cr-ster 10, the rRNA genes are

normally present s taninly repeated copies located

within the nucleolar oganizer regions of the chromo-

somes. D. Brown, J. Gardon, and tolleagues have

shown that there are bout 500 copies of the rRNA

gene in each of the two mucleolar organizer regions of

diploid nuclei of Xenopus laevis. The rRNA precursors

are synthesized and processed in the nucleoli

Given that about 1030 rRNA genes evisi per dip

loic nucleus, it has

estimated that over 4

ould be required to saathesize the large number of

rRNA molecules preser in mature Xeoos eggs

ardl a plausible situation given the average lire ex

pectancy of a toad Ihis potential dilemma has been

resolved by the evolutionof a mechanism by%hich the

a plausible situation given the

life
GENE TRANSCRIPT POPULATIONS ARE

DIVERGENT IN DIFFERENT CELL TYPES

In higher eukaryotes, only a sınall proportion of the

genome s represenied among mkNA motecuies in any

eiven cell type. s has been demonstrated by RNA-

DNA saturation bybridization experiments RNA is

extracted from cells ofa paticular type and allowed to

hybridize with total nuclear DNA (denatured).

RNA-DNA saturation hybridization experiments

have been done on a number of eukaryoxic species

ustng RNA from several different cell types. The results

of these experiments show that less than 10 percentoj

tbe DNA in the genome is represented by mRNA

molecules in the cytoplasm of any one cel ype lo

mice, for example, from 2 to 5 percent of the DNA

sequences are represented in the mRNA molecules

přesenr in liver cells. Brain cells appear to contain the

maximum variety of RNA transcripts. In the toad Xeno-

pus, 8 percent of the DNA sequences are represented

in mRNA from brain cells. The mRNA from Xenopus

oöcytes, on the othe. hand, contains sequences homol-

ogous to Tess than I percent of the DNA sequences It

is tear thererfore, thar the majority of the DNA s

quences in the genome

represented among the mRNA populations of

one tissue.or cell type

Since over 90 percentofthe DNA sequences in the

genome art nof represented

among mRNA popula

rons in any given cell, it has been lypethesiz

state, and that the regulation of transcription andor

tivaing the transcpption of speciti f

eukaryotuc gertes are packag

Chapter 6, pp. 136-143) in a nonspecifically repressed

jn (see ;

transcript processing occurs by a positive mechanism

Involving speaific gene activators These activators are

proposed to function by somehow turning on or ac

ic genes, or sets o

els. What

enes, at the proper time in the appropriate

these activators are, and how such activation might

occur are just beginning to be established. In several

cases, the activation is known to involve cis-acting

regulatory sequences called enhancers. The structure

and effects of enhancers are discussed in subsequen

sections of this chapter. Evidence indicates that certain

nonhistone chromosomal proteins function as specific

activators of transcription in some cases. Other evi

dence suggests that the regulation of RNA transcripo

processing is important in controlling differentiation

in eukaryotes. .

The histones are thought to beresponsible, at least

for the nonspecific repression of eukacyatic

genes. The histones, excluding histone H1.have been

highly conserved throughout the evolution of eu

ouc organisms

, and these histones are known to

igity complexed with rhe DNA in nucleosomes

However, RNA płymerase II of eukaryotes

which transcribes mos of the prorein encoding, nu

transcription accurately in

oteins

T reirement for

(Fig. 15.19) provides the

clear genes, cannot intne

ttro without the a

these transcription fac

potential tor additional s

of transcr

Most Eukaryotic Transcription Units

Are Monogenic
At present, our knowledge about the mechanisms

which gene expression

is regulated in euka

expanding rapidly. We know that different sets of

genes are transcribed in different cell types in higher

we know that the different pattems of

gene expression in diferent tissues are controlled by

trans-acing proteins encoded by regulatory genes that

quence during differentiation. Clearly, regula-

tory mechanisms acting at the level of transcription are

important in cell differentiation. However, the molec-

ular details of these regulatory mechanisms are still

being worked out, and many important questions

about differentiation promise to challenge geneticists

for years to come..

In higher eukaryotes,it does seem yery clear that

operons are net important if they exist at all. Althoug)h

ence for operons or operonlike units in

es teg, fungi), operons appear to

be rare or nonexistent in higher eukaryotes. Most of

the mRNAS of higher eukaryotes characterized to date

Enhancers and Silencers Modulate

Transcription in Eukaryotes

Eukaryotic genes are regulated by promoter elemen

located just upstream (5') from the transcripton-n

atic1 Sites in a manner quite simiłar to the regulatio

of prokaryotic genes (Chapter 14). However, as in t

case of the Drosopbila Ubx gene (see Fig 15.16), the

eukaryotic promoters may be very complex with bin

es for many different regulatory proteins.

tion to the nearby promoters, many eukaryo

genes are also regulated by more distant cis-actir

elements called enhancers and silencers As the nam

suggest, enhancers increase transcription and silen

ors decrease transcription of the regulated gene

Since enhancers appear to be more common and a

much

focus on the properties of enhancer elements

better understood, the following discussion w

The basic features of enhancers that distinguis

them from promoters are as follows

1 Enbancers can act over relatively Large distances

to several thousand nucleotide-pairs from the regula

gene(s)

Enbancers are orientation independent- they func

tion equally well in either orientation, normal or in

verted (tu ned end-for end)

3. Enbancers are position independent- they function

equally we!! whether located upstream (5') from a gende,

downstream (3') from a gene, or present withirn

intron of a gene.

The most extensively studied enhancer is tha

present on the minichromosome

of simian virus 4

(SV40), a virus of monkeys that can be investigated ir

celi culures. The complete SV40 enhancer is abou

220 nucleotide-pairs in length (Fig, 15.20). This region

of the 5V40 minichromosome is not packaged into

mably, the SV40 en

hancer is covered with protein transcription factors

that prevent it from becoming wrapped into nucleo-

somes by the histones. The SV40 enhancer contains

two 72-nucleotide-pais direct repeats, and delction of

both repeats eliminates cnhancer activity. If one of the

nhancet is still func-

nal.

Models of enhancer and silencer action

thus resemble the mechaism of regulaiion of the ara

operoninE cOLE (see Chapter 14, Fig 14.8), e

many more regulatory

in higher eukaryotes Far example, in the case of the

chicken P

implicatés the bind

to the promoter and at East five different proteins to

the ennancer.
Regulation of levels of transcription by DNA methylation.

lo date, there has been no definitive proof of the

role of methylation in the regulation of the expression

of any eukaryouc gene. Instead, the arguments for the

involvement of methylation in the control of gene

expression in e

d primarily on three

umerous studies nave

demonstrated a correlation between the

expression and the degree of methylation, such that

low methylation high gene expression, high meth-

ylation-low gene expression. (2) Methylation pat-

terns are tissue specific, at least in some cases. (3) The

drug (base analog) 5-azacyüdine, which can not be

methylated after it is Incorporated into DNA has.been

shown to result in the expression of genes in tis ues

where they normally are no

ne

ted DNA One of the more interesting discoveries of the last

set decade was that segments of DNA that have sequences

in which purines and pyrimidines alternate along each

ession strand can form left-banded double helices. The Wat

nt cell son-Crick B-form of DNA is a right-banded double

up on helix. The novel ieft-handed double-helical form of

ーC

ogmly DNA has been named Z.DNA for the zigzagged

hosphate backbones of. the molecule

c pa

pecific (Fig. 15.24)

das a

he Z-form of altemating purine and

pyrimidine DNA sequences occurs only at high salt

concentrations. When some of the bases in the poten

tial 2-form sequences are methylated (see the preced

ing section), however, the Zconformation is stable at

lower salt concentrations. Thus, Z-DNA composed of

altemating purine pyrimidine sequences containing

subse-

ethylated bases may be stable in vivo. Moreover, its

stability is enhanced by cations, including polyamines

uch as spermine, by negative supercoiling (see Chap-

el, we ter 5, pp. 124-126), and by DNA binding proteins

l type s

osuch specific for Z-DNA.

tified

In fact, there is evidence that Z-DNA exists in the

tidine interband regions of the giant salivay gland Chromo-

e fetal somes of Dro

YOniC SC
melanogaster and in the, tran

yonic sripuonatly active macronucleus of the ciliated proto-

prepared anibodies specific for ZDNA these antibodies do not react with B-form DNA.

When , alternating purine-pyrimidine sequences are complexed with histones, these sequences do not
display B-DNA to Z-DNA transitions. Z-DNA sequences may not be found in nucleosomes.

Experiments designed to test the

possibilities (1) that B-form to Z-form transitions in

DNA are involved in the regulation. ofgene expression

and (2) that regulatory proteins may act by binding to

and stabilizing one or the other ofthese conformations

may lead to some exciting deveiópments during the

next tew years