1.
Internal organization of Nucleus
and Nucleolus
- Himanshu Upadhyay
MSc. MLT Clinical Biochemistry

2.
Introduction
 The nucleus is more than a container in which chromatin, RNAs and nuclear
proteins move freely in aqueous solution.
 Discovered in 1831 by Scottish botanist Robert Brown. He suggested the nucleus
played a key role in fertilization and development of the embryo in the plants.
 The nucleus appears to have an internal structure that organizes the genetic
material and localizes some nuclear functions to discrete sites.
 The most recognizable aspect of the internal organization of the nucleus is the
nucleolus, which is the site at which the rRNA genes are transcribed and
ribosomal subunits are assembled.
 Additional elements of internal nuclear structure are suggested by the
organization of chromosomes and by the potential localization of functions such
as DNA replication and pre-mRNA processing to distinct nuclear domains.
Himanshu Upadhyay (MSc. Clinical Biochemist.)
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3.
Chromosomes and Higher-Order Chromatin Structure
 Chromatin becomes highly condensed during mitosis to form the compact metaphase
chromosomes that are distributed to daughter nuclei. During interphase, some of the
chromatin (heterochromatin) remains highly condensed and is transcriptionally
inactive; the remainder of the chromatin (euchromatin) is decondensed and
distributed throughout the nucleus.
 Cells contain two types of heterochromatin. Constitutive heterochromatin contains
DNA sequences that are never transcribed, such as the satellite sequences present at
centromeres.
 Facultative heterochromatin contains sequences that are not transcribed in the cell
being examined, but are transcribed in other cell types. Consequently, the amount of
facultative heterochromatin varies depending on the transcriptional activity of the
cell.
 Much of the heterochromatin is localized to the periphery of the nucleus, possibly
because one of the principal proteins associated with heterochromatin binds to a
protein of the inner nuclear membrane.
Himanshu Upadhyay (MSc. Clinical Biochemist.)
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4.
Diagrammatic representation of Heterochromatin in interphase
nucleiHimanshu Upadhyay (MSc. Clinical Biochemist.)
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5.
 Although interphase chromatin appears to be uniformly distributed, the chromosomes are
actually arranged in an organized fashion and divided into discrete functional domains
that play an important role in regulating gene expression.
 The nonrandom distribution of chromatin within the interphase nucleus was first
suggested in 1885 by C. Rabl, who proposed that each chromosome occupies a distinct
territory, with centromeres and telomeres attached to opposite sides of the nuclear
envelope.
Himanshu Upadhyay (MSc. Clinical Biochemist.)
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6.
 This basic model of chromosome organization was confirmed nearly a hundred
years later (in 1984) by detailed studies of polytene chromosomes in Drosophila
salivary glands. Rather than randomly winding around one another, each
chromosome was found to occupy a discrete region of the nucleus.
 This diagram represent organization of Drosophila
Chromosomes.(A) A model of nucleus, showing the
five chromosomes arms in different colors, the
telomers and centromeres are indicated. (B)The two
arms of chromosomes are shown to illustrate the
topological separation between chromosomes.
 The chromosomes are closely associated with the nuclear envelope at many
sites, with their centromeres and telomeres clustered at opposite poles.
Himanshu Upadhyay (MSc. Clinical Biochemist.)
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7.
 The chromatin in interphase nuclei appears to be organized into looped domains
containing approximately 50 to 100 kb of DNA. A good example of this looped-domain
organization is provided by the highly transcribed chromosomes of amphibian oocytes, in
which actively transcribed regions of DNA can be visualized as extended loops of
decondensed chromatin.
 These chromatin domains appear to represent discrete functional units, which
independently regulate gene expression.
 This diagram represent Looped chromatin domains. Light micrograph of a chromosome of
amphibian oocytes, showing decondensed loops of actively transcribed chromatin
extending from an axis of highly condensed non-transcribed chromatin.
Himanshu Upadhyay (MSc. Clinical Biochemist.)
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8.
Functional Domains within the Nucleus
 Have distinct region for various processes.
 Contains multiple clustered site for DNA replication.
 Speckles: storage site for splicing components.
 Promyelocytic leukaemia protein (PML) bodies : site for transcriptionally
regulatory proteins involved in acute promyelocytic leukaemia.
 Cajal Bodies: enriched in small Ribonucleoproteins (RNPs), function as site of
RNP assembly and processing.
 Nucleolus : site for ribosome synthesis.
Himanshu Upadhyay (MSc. Clinical Biochemist.)
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9.
Nucleolus
 Largest structure present inside the boundaries of the nucleus.
 Dark staining zone in centre of nucleus.
 Sit of rRNA transcription and processing.
 Main components are RNA , DNA and proteins
 Known as ribosome production factory.
Himanshu Upadhyay (MSc. Clinical Biochemist.)
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10.
Ribosomal rRNA genes
 Nucleolus has gene 5.8s, 18s and 28s rRNA.
 Ribosomal rRNA genes are present in tandem array.
 Around 200copies of gene coding for 5.8s, 18s, 28s rRNA present on chromosome
13, 14, 15, 21 and 22.
 5s rRNA gene is present outside nucleolus on chromosome 1 in a tandem array.
 5.s, 18s and 28s are transcribed as single unit by RNA polymerase 1, yielding 45s
pre-rRNA.
 Pre-rRNA is processed to 18s rRNA of 40s subunit and 5.8s, 28s rRNA of 60s
subunit of ribosomes.
Himanshu Upadhyay (MSc. Clinical Biochemist.)
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11.
Organization of the Nucleolus
 The importance of ribosomal production is particularly evident in oocytes in
which the rRNA genes are amplified to support the synthesis of the large
numbers of ribosomes required for early embryonic development.
 In xenopus oocytes, the rRNA genes are amplified approximately 2000 fold,
resulting in about 1 million copies per cell.
 These amplified rRNA genes are distributed to thousands of nucleoli which
support the accumulation of 1012 ribosomes per oocyte.
 Morphologically, Nucleoli has 3 regions i.e. fibrillar centre, dense fibrillar
component and granular component.
 These regions represent the sites of progressive stages of rRNA transcription,
processing and ribosome assembly.
Himanshu Upadhyay (MSc. Clinical Biochemist.)
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12.
 Nucleoli become associated with the chromosomal regions that contains the 5.8s,
18s, 28s rRNA genes, which are called as nucleolar organizing regions.
 Formation of nucleoli requires the transcription of 45s pre-rRNA.
 The size of nucleolus depends on the metabolic activity of the cell.
 Large nucleoli fund in the cells that are actively engaged in protein synthesis.
Himanshu Upadhyay (MSc. Clinical Biochemist.)
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13.
Transcription and Processing of rRNA
 Each nucleolar organizing region contain a cluster of tandemly repeated rRNA
genes separated from each other by non-transcribed spacer DNA.
 These genes are very actively transcribed by RNA polymerase 1.
Himanshu Upadhyay (MSc. Clinical Biochemist.)
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14.
Ribosome Assembly
 Ribosomal proteins are imported
to the nucleolus from the
cytoplasm and begin to assemble
on pre- rRNA prior to its cleavage.
 As the pre-rRNA is processed,
additional ribosomal proteins and
the 5s rRNA (which is synthesizes
elsewhere in the nucleus)
assemble to form pre-ribosomal
particles.
 The final steps of malnutrition
follow the export of pre-ribosomal
particles to the cytoplasm,
yielding the 40s and 60s ribosomal
subunits.
Himanshu Upadhyay (MSc. Clinical Biochemist.)
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15.
Himanshu Upadhyay (MSc. Clinical Biochemist.)
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