Download An Introduction to Genetic Analysis Chapter21 Extranuclear Genes

An Introduction to Genetic Analysis
Chapter21
Extranuclear Genes
Chapter21
Extranuclear Genes
Key Concepts
Chloroplasts and mitochondria each contain multiple copies of their own unique
“chromosome” of genes.
Generally, organelle DNA—and any variant phenotype encoded therein—is inherited through
the maternal parent in a cross.
In mixtures of two genetically different mitochondrial DNAs or chloroplast DNAs, it is
commonly observed that a sorting-out process results in descendant cells of one type or the
other.
In “dihybrid” organelle mixtures, recombination can be detected.
Organelle genes encode mainly organelle translation components and components of
energy-producing systems.
Most organelle-encoded polypeptides unite with nucleus-encoded polypeptides to produce
active proteins, which function in the organelle.
Introduction
By far the larger proportion of the DNA of eukaryotic organisms is found in the nuclear
chromosomes. However, two types of organelles, mitochondria and chloroplasts (Figure 21-1),
each contain a unique type of “chromosome” of genes that encode specific functions of that
organelle. The mitochondrial chromosome is called mtDNA, and the chloroplast chromosome
is cpDNA. The functions of mitochondrial genes are directed at making ATP (“chemical
energy”) by oxidative phosphorylation, which takes place in the mitochondrion itself.
Chloroplast genes are ultimately concerned with making ATP by photosynthesis.
The number of genes in organellar chromosomes is small relative to the number in the nucleus.
For example, the human nuclear genome consists of 3,000,000 kb of DNA containing about
100,000 genes, whereas human mtDNA is only 17 kb and has only 37 genes. In any one
organism, a gene found in an organelle chromosome is generally not found in the nuclear
chromosomes, although a few may be present in the nucleus as inactive pseudogenes. In
structure and function, organelle genes show many generic similarities with nuclear genes, but
there are enough differences in their action and inheritance to make them worthy of
specialized treatment in a chapter of their own.
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MESSAGE
Mitochondria and chloroplasts each contain a relatively small unique type of
chromosome containing some of the genes needed for the function of that organelle.
Origin of extranuclear genes
The question of how the mitochondria and chloroplasts came to have these specific sets of
genes is still a matter of experimentation and debate in biology. Part of the answer is to be
found in the origin of the chloroplasts and mitochondria themselves. It is generally believed
that these two organelles arose in the course of evolution as endosymbionts. Specifically, cells
of ancestors of eukaryotes were “invaded” at different times by prokaryotic cells, one of which
was photosynthetic, giving rise to chloroplasts, and one of which was nonphotosynthetic,
giving rise to mitochondria. These invasions set up mutually beneficial symbioses. This
symbiosis was a key event in the origin of the lines that eventually became modern
eukaryotes.
However, the ancestral invading prokaryotes must have contained many more genes than are
found in modern mitochondria and chloroplasts. The evidence suggests that some of these
genes were lost, whereas others found their way into the nucleus. The precise set of genes that
remains in the organelles of modern eukaryotes is somewhat variable, although a core set
tends to be found in most organisms. It is likely that there is adaptive advantage in having
some organelle genes located in the organelle itself. The differences between organisms
presumably are due to different migration patterns of the organelle genes in the evolution of
different eukaryotes. The precise reasons for the differences are not known.
Most modern eukaryotic cells are fully dependent on the organelle genes for their normal
function; hence what arose originally as an optional symbiosis is now obligatory. Nevertheless,
it is known that some organisms can survive without their mitochondria or their chloroplasts.
For example, the yeast Saccharomyces cerevisiae can obtain energy from fermentation, a type
of chemistry that does not need the mitochondrial genes. Hence mutants that lack
mitochondrial genes survive. In another example, some plants can survive saprophytically
without their chloroplast genes.
Structure of organelle chromosomes
Mitochondria and chloroplasts can be isolated by various cell fractionation methods, and from
these fractions organelle DNA is isolated in the usual way. With the use of standard
recombinant DNA technology (Chapters 12 and 13), several organelle chromosomes have now
been fully sequenced. The functions of the organelle genes have been determined by a
combination of mutation analysis (see later section) and homology with DNA databank
sequences of known function.
Overall organization
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Here we see the first big difference between organelle and nuclear chromosomes. Most
organelle chromosomes appear to be fundamentally circular. The evidence for their circularity
is that restrictions maps of organelle DNA are circular, and furthermore DNA circles can be
seen in organelle preparations under the electron microscope. There is evidence that some
organelle chromosomes can take on linear forms, but by and large most geneticists treat them
as though they are circles.
Another important difference in overall organization is that organelle chromosomes are not in
the highly condensed form found in eukaryotic chromosomes; that is, they are not in a
euchromatic state.
How many copies?
Here we see another difference: whereas nuclear chromosomes are present as either one copy
per cell (haploid) or two copies (diploid), organelle chromosomes are present in many copies
per cell, often in the hundreds or thousands. The regulation of this cellular copy number is
relatively loose; so in different cells of the same organism there is some variation around a
mean value.
The leaf cells of the garden beet have about 40 chloroplasts per cell. The chloroplasts
themselves contain specific areas that stain heavily with DNA stains; these areas are called
nucleoids, and they are a feature commonly found in many organelles. Each beet chloroplast
contains from 4 to 8 nucleoids, and each nucleoid contains from 4 to 18 cpDNA molecules.
Thus, single cells of a beet leaf can contain as many as 40 × 8 × 18 = 5760 copies of the
chloroplast genome. Although the photosynthetic protist Chlamydomonas has only one
chloroplast per cell, the chloroplast contains from 500 to 1500 cpDNA molecules, commonly
observed to be packed in nucleoids.
What about mitochondria? A “typical” haploid yeast cell can contain from 1 to 45
mitochondria, each having from 10 to 30 nucleoids, with 4 or 5 molecules in each nucleoid.
The mitochondrial nucleoids of the unicellular Euglena gracilis are shown in Figure 21-2. In
human cells, there can be from 2 to 10 mtDNA molecules per mitochondrion. The number of
mitochondria per cell is different in different cell types. Hence both number per mitochondrion
and number of mitochondria vary. There are several hundred mtDNA molecules in human
fibroblast cells and approximately 100,000 in human oocytes.
Mitochondrial genomes
The yeast and human mitochondrial genomes are shown in Figure 21-3. This view of the
mitochondrial genome shows it to have two main functions: (1) it encodes some of the
proteins that constitute the oxidative phosphorylation system and (2) it encodes tRNAs,
rRNAs, and some proteins used in mitochondrial protein synthesis.
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Yet it is striking that not all the components of the oxidative phosphorylation system are
encoded in the mtDNA. The remaining proteins are encoded by nuclear genes, and the mRNA
is translated outside the mitochondrion on cytosolic ribosomes. Proteins made on these
cytosolic ribosomes are transported into the mitochondrion, and the complete system is
assembled in the mitochondrial inner membrane (Figure 21-4).
Genes for 25 yeast and 22 human mitochondrial tRNAs are shown on the maps in Figure 21-3.
These tRNAs carry out all the translation that takes place in mitochondria. They are far fewer
than the minimum of 32 required to translate nucleus-derived mRNA. The economy is
achieved by a “more wobbly” wobble pairing of the tRNA anticodons (see Chapter 10). The
tRNA specificities in human mtDNA are shown in Figure 21-5. Notice that the codon
assignments are in some cases different from those of the nuclear code. The code also varies in
different species. Hence, the genetic code is not universal, as had been supposed for many
years.
The map contains some other surprises. Most prominent are the introns in several
mitochondrial genes of yeast. Subunit I of cytochrome oxidase contains nine introns. The
discovery of introns in the mitochondrial genes of yeast is particularly surprising because they
are relatively rare in yeast nuclear genes. Another surprise is the existence of unassigned
reading frames (URFs) within the yeast introns. URFs are sequences that have correct
initiation codons and are uninterrupted by stop codons. Some URFs within introns appear to
specify proteins important in the splicing out of the introns themselves at the RNA level.
Notice that human mtDNA is by comparison much smaller and more compact than yeast
mtDNA. There is much less spacer DNA between the genes.
MESSAGE
Mitochondria contain multiple copies of mtDNA, a circular molecule with genes for
mitochondrial protein synthesis (mainly rRNAs and tRNAs) and for subunits of the
proteins associated with mitochondrial ATP production. Less understood regions
include the introns, unassigned reading frames, and spacer DNA.
Chloroplast genomes
Figure 21-6 shows the organization and functions of most of the genes of the cpDNA from the
liverwort Marchantia polymorpha. Typically, cpDNA molecules range from 120 to 200 kb in
different plant species. In Marchantia, the molecular size is 121 kb.
The Marchantia molecule contains about 136 genes, including those encoding four kinds of
rRNA, 31 kinds of tRNA, and about 90 proteins. Of the 90 protein-encoding genes, 20 encode
photosynthesis and electron-transport functions. Genes encoding translational functions take
up about half the chloroplast genome and include those encoding the proteins and RNA types
necessary for translation in the organelle.
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Notice the presence of a large inverted repeat in Figure 21-6. Such inverted repeats are found
in the cpDNA of virtually all species of plants. However, there is some variation in regard to
which genes are included in the inverted repeat region and therefore in the relative size of that
region. One of the mysteries of the inverted repeat is that the duplicates have exactly the same
sequence, yet to date no mechanism is known that ensures this complete identity.
Like mtDNA, cpDNA cooperates with nuclear DNA to provide subunits for functional
proteins used inside the organelle. The nuclear components are translated outside in the
cytosol and then transported into the chloroplast, where they are assembled together with the
components synthesized in the organelle.
MESSAGE
Chloroplasts contain multiple copies of cpDNA, a circular molecule that contains
genes for photosynthesis, electron transport, and chloroplast protein synthesis. Most
cpDNAs contain an inverted repeat.
Organelle mutations
Like nuclear genes, the organelle genes are mutable. Indeed, in mammalian mtDNA, the
base-pair-substitution rate is approximately 10 times as high as that of nuclear genes. (Plant
organelle DNA does not show these high rates.) Deletions and other rearrangements also are
found.
Many of these DNA changes are expressed as abnormal phenotypes at the cellular and
organismal level. Because organelles produce energy, a typical mutant phenotype is energy
deficiency and hence slow growth or sickly appearance. Mutations in the genes for
electron-transport components are often of this type. Mutations in rRNA- or ribosomal
protein-encoding genes often lead to resistance to specific drugs such as streptomycin and
erythromycin, antibiotics whose effect is exerted by binding to ribosomes. Plant mutations in
chloroplast DNA sometimes lead to a white phenotype, indicating a lack of the green
photosynthetic pigment chlorophyll. Some examples of mitochondrial and chloroplast mutants
follow.
One of the first mitochondrial mutants discovered was a slow-growing mutant of the fungus
Neurospora. Because of its slow growth, this mutant became known as poky. Although at the
time of its discovery the mitochondrial basis of poky was inferred from its inheritance pattern
(see next section), it is now known to be a deletion of four base pairs in the gene for the small
subunit of mitochondrial rRNA. Because Neurospora is an obligate aerobe, it cannot survive
without functional mitochondria; so none of the Neurospora mutants are nulls, and they retain
some function.
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In budding yeast, point mutations in some electron-transport proteins cause a slow rate of cell
division resulting in small colonies (mit mutations—see Figure 21-3 for their location).
Deletions of part or even all of the mtDNA also produce small colonies (called petites). Yeast
cells can obtain ATP by fermentation, which does not rely on the mitochondrial oxidative
phosphorylation system, so yeast with these drastically deleted genotypes can survive, albeit at
a reduced activity level. Petites in which part of the mtDNA has been deleted regenerate
full-sized mtDNA molecules, as shown in Figure 21-7.
The photosynthetic protist Chlamydomonas reinhardtii has been the subject of extensive
analysis of mutations in the cpDNA, starting with the work of Ruth Sager in 1954. Sager
isolated a large number of antibiotic-resistant and other abnormal phenotypes and, by
correlating their unusual inheritance patterns with those of cpDNA, showed that they were
almost certainly mutations in cpDNA itself.
In humans, several diseases have been shown to be caused by mutations in mtDNA. In general
these diseases are called mitochondrial cytopathies. The organs most affected by these
mutations are those in which there is a high energy demand, notably muscles and nerves. The
mutations are either point mutations in individual mitochondrial genes or large deletions. The
positions of some of these mutations are shown in Figure 21-8. The common deletions almost
certainly arise from a crossover between direct repeats (Figure 21-9). (This same mechanism
has also been detected in fungal mtDNA and plant cpDNA, where both the large and small
circular products have been found.)
Myoclonic epilepsy and ragged red fiber (MERRF) disease is an example of a human disease
resulting from a mitochondrial point mutation—in this case, nearly always a substitution of G
for A at position 8344 in the tRNA gene for lysine. It is a muscle disease (myopathy), but
symptoms also include eye and hearing disorders. The ragged red muscle fibers show absence
of oxidative phosphorylation.
Kearns-Sayre (KS) syndrome is a constellation of symptoms affecting eyes, muscles, heart,
and brain. It is nearly always associated with a deletion in mtDNA.
Mutations in the nuclear components of the oxidative phosphorylation system of mitochondria
and the photosynthetic complexes of chloroplasts result in many of the same types of
phenotypic expression as those of their organelle-encoded counterparts. For example,
chlorophyll-less mutants of plants are often caused by defective nuclear genes that encode
some aspect of chlorophyll structure or function (Figure 21-10). These mutations, however,
are inherited in a strict Mendelian manner, as expected of nuclear genes, not in the
non-Mendelian manner to be described in the next section.
MESSAGE
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Organelle mutations can result in abnormal growth, abnormal amounts of organelle
proteins, defective electron transport, antibiotic resistance, and (in cpDNA) abnormal
photosynthesis.
Inheritance of organelle genes and mutations
We shall consider inheritance at three levels—expression, cytoplasmic segregation, and
maternal inheritance (Figure 21-11). First, because a cell contains many copies of organelle
DNA, it is intuitively difficult to see how a mutation affecting expression can rise to a position
where it will influence the phenotype of the cell and the organism. Yet we must remember that
organelle DNA replicates even in cells that are no longer dividing. We shall consider some
possible fates of mutant organelle DNAs within a cell.
Second, a cell in which both wild-type and mutant organelle DNA coexist is called a
heteroplasmon or, sometimes, a cytohet. When heteroplasmons divide asexually, daughter
cells are commonly observed to contain only one or the other organelle DNA type. This type
of inheritance is called cytoplasmic segregation. We shall consider how this segregation
might take place.
The third level of inheritance concerns transmission during the sexual cycle. Organelles are
located in the cytoplasm, so it is expected that they will show an inheritance pattern
characteristic of that location. In the zygote of an organism that is heterogametic (has
different-sized male and female gametes), virtually all the cytoplasm is derived from the egg
of the mother. Hence we expect the organelles, organelle DNA, and organelle mutations to
follow this cytoplasmic line of descent. This type of inheritance is called maternal inheritance.
These three processes are now considered in more detail.
Expression of organelle mutations
How can a mutation rise from a frequency of 1 in several hundred or several thousand to a
state in which it can express itself in the phenotype? There are at least three hypotheses that
account for this rise.
First, some organelle mutations are suppressive. This word does not mean that they act as
suppressors (see Chapter 4). Suppressivity means that they can outreplicate the wild-type
organelle genomes within a cell. This renegade activity of mitochondrial mutations is
unexpected; after all, our experience with nuclear genes is that mutants either die or stay at a
low frequency. However, it seems to be a characteristic of certain types of mutant organelle
genomes that they can gain ascendancy in the cell in which they arise. A second possibility,
for which there is experimental support, is that the frequency of mtDNA types can rise and fall
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entirely on the basis of chance (called random drift). Sometimes the frequency drifts so far
that one mtDNA type is completely eliminated. A third possibility is that mitochondria
containing certain types of mutations have a mechanism for recognizing that there is a
potential energy deficiency and start replicating faster.
Let's look at some examples of suppressive organelle mutations. The first example ever found
was a class of petite mutations in budding yeast called suppressive petites. When petite cells of
this type are fused with wild-type cells to make a heteroplasmon, the petite-causing
mitochondria gain ascendency in the mixture and most of the subsequent cells are generally
petite in phenotype. These petite mutations are the deleted type in which the remaining
fragment is tandemly repeated. It is likely that the cause of the suppressivity is that replication
origins are duplicated.
In Neurospora, the mitochondrial mutation abn (abnormal) has the same effect. If cells of this
type are fused with wild-type cells even at very lopsided fusion ratios (say 10,000:1 in favor of
wild type), the heteroplasmic mycelium (which is really one big cell) quite rapidly expresses
the mutant abnormal phenotype, and the underlying mtDNA becomes predominant. The same
effect can be observed by injecting a small number of abn mitochondria into a wild-type cell.
In humans, a parallel situation is found. The frequency of mutant mtDNA forms associated
with the mitochondrial cytopathies often changes throughout life, and different parts of the
body contain different proportions. This situation is especially pronounced in cells that are
postmitotic (that is, will never again undergo mitosis). For example, in one patient an mtDNA
deletion was found in the following proportions:
There is evidence of a threshold effect for some cytopathies; when the frequency of the
mutant type rises above this threshold level, the disease symptoms are expressed. Localized
high levels of mutant mtDNA can severely disrupt function of organs such as muscle.
Cytoplasmic segregation
The term cytoplasmic segregation is used to describe the production of mutant and wild-type
descendant cells of a heteroplasmon. Some of the mechanisms proposed for mutant expression
in the preceding section also pertain to cytoplasmic segregation. For example, if a mutant type
can drift to ascendency in one part of a heteroplasmon, it seems likely that this area can give
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rise to mutant descendant cells.
Cytoplasmic segregation has been used as the basis for the heterokaryon test for mitochondrial
mutations in filamentous fungi. If a new mutation has arisen and is suspected of being
mitochondrial, a heterokaryon is forced with a wild-type strain. (Forcing means that both
components must carry a nuclear auxotrophic marker that prevents growth as a separate
strain.) In most fungi, the nuclei never or rarely fuse; so, if cells bearing the nuclear
auxotrophic marker of the wild-type partner can be recovered with the new mutant phenotype,
that nucleus has likely acquired the mutant phenotype by cytoplasmic contact and subsequent
cytoplasmic segregation. Hence the mutation is most likely mitochondrial. Figure 21-12 shows
this process.
MESSAGE
When a heteroplasmic cell divides, cytoplasmic segregation can occur to produce
daughter cells with one organelle DNA or the other.
Maternal inheritance
This type of inheritance pattern can be illustrated by several landmark crosses in the study of
mitochondrial genomes.
Maternal inheritance of poky
Neurospora.
In 1952, Mary Mitchell isolated the poky mutant strain of Neurospora. This
mutant differs from the wild-type fungus in a number of ways: it is slow growing (as
mentioned earlier), and it has abnormal amounts of mitochondrial cytochrome proteins. Like
most organisms, wild-type Neurospora has three main types of cytochrome: a, b, and c . Poky,
however, lacks cytochromes a and b and has an excess of cytochrome c.
Mitchell established the cytoplasmic basis for the poky mutation by showing that it was
inherited maternally. It is possible to cross some fungi in such a way that one parent
contributes the bulk of the cytoplasm to the progeny; this cytoplasm-contributing parent is
called the maternal parent, even though no genuine male or female parents exist. Mitchell
demonstrated maternal inheritance for the poky phenotype in the following reciprocal crosses:
In such crosses, any nuclear genes that differed between the parental strains were observed to
segregate in the normal Mendelian manner and to produce 1:1 ratios in the progeny (Figure
21-13). All poky progeny behaved like the original poky strain, transmitting the poky
phenotype down through many generations when crossed as females.
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Maternal inheritance of chloroplast pigments in plants.
In 1909, Carl Correns reported some surprising results from his studies on variegated
four-o'clock plants (Mirabilis jalapa). He noted that most of the leaves of these variegated
plants show patches of green and white tissue, but some branches carry only green leaves and
others carry only white leaves (Figure 21-14).
Flowers appeared on all types of branches, so Correns intercrossed a variety of different
combinations by transferring pollen from one flower to another. Table 21-1 shows the results
of such crosses. Two features of these results are relevant. First, there is a difference between
reciprocal crosses: for example, white female × green male gives a different result from green
female × white male. Overall, the phenotype of the maternal parent is solely responsible for
determining the phenotype of all progeny. The phenotype of the male parent appears to be
irrelevant, and its contribution to the progeny appears to be zero. This is a case of strict
maternal inheritance. Although the white progeny plants do not live long, because they lack
chlorophyll, the other progeny types do survive and can be used in further generations of
crosses. In these subsequent generations, maternal inheritance always appears in the same
patterns as those observed in the original crosses.
Figure 21-15 diagrams a model that formally accounts for all the inheritance patterns in Table
21-1. Variegated branches apparently produce three kinds of eggs: some contain only white
chloroplasts, some contain only green chloroplasts, and some contain both kinds. The egg type
containing both green and white chloroplasts produces a zygote that also contains both kinds
of chloroplasts. In subsequent mitotic divisions, the white and green chloroplasts segregate in
some cell lines, thus producing the variegated phenotype. Here, again, we see the phenomenon
of cytoplasmic segregation.
In most plants, organelles are inherited from the maternal parent. However there are some
glaring exceptions in which inheritance is strictly paternal.
Uniparental inheritance in
Chlamydomonas reinhardtii.
The life cycle of Chlamydomonas is shown in Figure 21-16.
In 1954, Ruth Sager isolated a streptomycin-sensitive mutant with an inheritance pattern that
at the time was highly unexpected. In the following crosses, sm-r and sm-s indicate
streptomycin resistance and sensitivity, respectively. The mating type gene is mat, with alleles
+ and −. (Crosses can take place only between + and − cultures.)
Here we see again a difference in reciprocal crosses; all progeny cells show the streptomycin
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phenotype of the mat+ parent. Like the maternal-inheritance phenomenon, this is a case of
uniparental inheritance. In fact, Sager referred to the mat+ mating type as the female, even
though there is no observable physical distinction between the mating types; nor is there a
difference in the contribution of cytoplasm as seen in Neurospora. In these crosses, the
conventional nuclear marker genes (such as mat itself) all behave in a Mendelian manner and
give 1:1 progeny ratios.
Several other mutants (referred to earlier) showed uniparental inheritance. These experiments
revealed to Sager the existence of a mysterious “uniparental genome” in
Chlamydomonas—that is, a group of genes that all show uniparental transmission only
through the mat+ parents in crosses. This uniparental genome is the chloroplast DNA
(cpDNA). In a zygote the cpDNA of the mat− parent was shown to be destroyed in some way.
This destruction can be demonstrated easily by showing that the restriction pattern of cpDNA
of the progeny is always the same as the mat+ parent. This loss of cpDNA from the mat−
parent parallels the loss of cpDNA genes (such as the sm genes) borne by the mat− parent.
In Chlamydomonas, the mtDNA and its mutations also are inherited uniparentally, but,
perversely, the mtDNA is inherited from the mat− parent. In other words, in a cross, all the
progeny carry the mtDNA genotype of the mat− parent.
Uniparental inheritance of mitochondrial mutations in budding yeast.
In a yeast cross, the two parental cells fuse and apparently contribute equally to the cytoplasm
of the resulting diploid cell (Figure 21-17). Hence a Neurospora-style “maternal” type of
inheritance based on unequal contribution of cytoplasm is not expected or observed.
Furthermore, the inheritance patterns of the mtDNA are independent of mating type. In this
sense, then, organelle inheritance in yeast is quite different from that of cpDNA mutations in
Chlamydomonas. However, a type of uniparental inheritance is shown, as the following
examples illustrate.
If a petite of the type that lacks mtDNA is crossed with wild type, none of the progeny are
petite. For this reason, these petites are called neutral petites. This is a type of uniparental
inheritance. However, suppressive petites do produce petite progeny in proportions that
correlate with the degree of suppressiveness.
In a sense, petites are quite atypical of mitochondrial mutations generally. The drug-resistance
and mit point mutations more clearly show the inheritance pattern of mitochondrial genomes
in this organism.
The inheritance of an erythromycin-resistant mutation is shown in Figure 21-18. The original
zygote is effectively a heteroplasmon, consisting of a mixture of the parental cytoplasms. In a
yeast cross, zygotes often divide mitotically as diploids before meiosis takes place. In the
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mitotic divisions, the two mtDNAs undergo cytoplasmic segregation, so the meiocytes are
“pure” in regard to their mtDNA type, and all spores in one tetrad will be either eryR or eryS.
Hence uniparental inheritance is shown at the level of individual meiocytes.
Maternal inheritance of human cytopathies.
Human mtDNA deletions tend to be de novo in origin and are not inherited maternally.
However, the various point mutations are inherited maternally. For example, the MERRF
mutation can be detected throughout a maternal lineage over several generations. However,
because of heteroplasmy, cytoplasmic segregation, and the threshold effect, the members of
the family may be severely affected, show only weak symptoms, or show no clinical
symptoms at all.
MESSAGE
Organelle DNA and associated phenotypes are inherited uniparentally, most often
through the maternal parent.
Recombination of extranuclear DNA
Recombination of mtDNA can take place in mitochondrially “dihybrid” heteroplasmons. It
must be through mitochondrial fusion and a crossing-over-like process, although few
molecular details of the process are known. In budding yeast, drug-resistance markers can be
used to demonstrate this process, as illustrated in Figure 21-19. The diagram shows that
recombination takes place in the zygote (heteroplasmon). When this cell divides mitotically,
cytoplasmic segregation results in tetrads that contain either one of the parental mtDNA
genotypes or one of the reciprocal recombinant types. The frequency of “recombinant tetrads”
was used historically to map mtDNA by using recombination units, but the technique had only
limited success and was largely supplanted by physical mapping techniques.
Recombination mapping has also been attempted in Chlamydomonas. Sager discovered that, if
the mat− parent is irradiated with UV light, its cpDNA is not inactivated and the zygote is a
heteroplasmon. Starting with such heteroplasmons, she was able to obtain recombinant
descendant cells, and their frequency in standardized procedures was used to partly map the
cpDNA.
Cytoplasmic male sterility
Male sterility in plants is often cytoplasmically based and maternally inherited. Male sterile
plants produce no functional pollen, but do produce viable eggs. Cytoplasmic male sterility is
used in agriculture to facilitate the production of hybrid seed. Hybrid seed is produced from a
cross between two genetically different lines; such seeds usually result in larger, more
vigorous plants. The main practical problem in producing hybrid seed is to prevent
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self-pollination, which would produce seeds that are not hybrid. One breeding scheme is
illustrated in Figure 21-20.
Mitochondria and aging
Among the theories of the mechanism of aging is the wear-and-tear theory. Cells are likened
to machines that over time accumulate damage that cannot be fully repaired and, in the end,
the machine cannot function and “dies.” Throughout the aging process of animals, there is a
reduction in oxidative phosphorylation, the function housed in mitochondria. Furthermore,
there is an accumulation of a certain deletion (the “common” 5-kb deletion) and certain point
mutations throughout aging. These kinds of observations have suggested the possibility of a
connection between mitochondrial mutation and mitochondrial wear-and-tear in aging. Indeed
aging might be the ultimate mitochondrial disease. There is some support for this theory. For
example, if the mtDNA of human cells is removed and replaced with mtDNA from people of
different ages, there is an agedependent correlation with oxidative phosphorylation in these
constructs.
In aging, the accumulation of mtDNA mutations does not seem to rise to a level sufficient to
interfere with oxidative phosphorylation. However, the effect of defective mtDNA molecules
at the cellular level is poorly understood. For example, if there were localized accumulation
within parts of a cell or in certain cells within a tissue, then the effect might be much greater
than the overall frequency would suggest. The mitochondrial theory of aging is still at the
speculative stage, and more research is needed on this topic.
Summary
Mitochondria contain multiple copies of a small circular “chromosome” of genes whose
functions relate to mitochondrial oxidative phosphorylation and to mitochondrial protein
synthesis. The set of mitochondrial genes does not provide an adequate set of proteins to carry
out these functions. The other necessary proteins are encoded in the nucleus, translated outside
the mitochondrion, and imported into the inner mitochondrial membrane. Chloroplasts also
contain many copies of a unique circular “chromosome” of DNA that contains genes mainly
related to photosynthesis and chloroplast protein synthesis. These genes also interact with
nuclear genes to become fully functional. Organelle translation uses a modified genetic code.
Mutations in some organelle genes lead to defects in the energy-producing systems and hence
slow or abnormal growth. Mutations in rRNAs and ribosomal proteins are often resistant to
specific drugs that bind to the ribosome.
To be expressed, organelle mutations must rise in frequency above an expression threshold.
Several mechanisms have been proposed that allow this to happen. In cells that are mixtures of
mutant and wild-type organelle DNA, cytoplasmic segregation leads to daughter cells of one
type or the other. When individuals carrying organelle mutations are used as parents in a
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sexual cross, the mutations are generally transmitted exclusively through the maternal parent.
In some organisms, there exists a specialized type of uniparental inheritance that is not
maternal in nature.
When two different organelle genotypes are combined in the same cell, a crossing-over type of
process can take place, leading to recombinant molecules. The frequency of such
recombinants has some use in mapping organelle chromosomes.
Solved Problems
1. In a strain of Chlamydomonas that carries the mt+ allele, a temperature-sensitive mutation
arises that renders cells unable to grow at higher temperatures. This mutant strain is crossed
to a wild-type stock, and all the progeny of both mating types are temperature sensitive.
What can you conclude about the mutation?
Solution
We are told that the mutation arose in a mat− stock. Therefore, the cross must have been
and the progeny must have been mat+ts and mat−ts. This is a clear-cut case of uniparental
inheritance from the mat+ parent to all the progeny. In Chlamydomonas, this type of
inheritance pattern is diagnostic of genes in chloroplast DNA, so the mutation must have
occurred in the chloroplast DNA.
2. Owing to evolutionary conservation, organelle DNA shows homology across a wide range
of organisms. Consequently, DNA probes derived from one organism often hybridize with
the DNA of other species. Two probes derived from the cpDNA and mtDNA of a fir tree are
hybridized to a Southern blot of the restriction digests of the cpDNA and mtDNA of two
pine trees, R and S, that had been used as parents in a cross. The autoradiograms follow
(numbers are in kb):
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The cross R♀ × S♂ is made, and 20 progeny are isolated. All are identical in regard to
their hybridization to the two probes. The autoradiogram for each progeny is:
a. Explain the probe hybridization of parents and progeny.
b. Explain the progeny results. Compare and contrast them with the results in this chapter.
c. What do you predict from the cross S♀ × R♂? Note: This problem is based on results
shown in several conifer species.
Solution
a. For both cpDNA and mtDNA, the total amount of DNA hybridized by the probe is
different in plants R and S. Hence, we can represent the DNA something like this (other
fragment arrangements are possible):
Thus, the probes reveal a (presumably neutral) restriction fragment length polymorphism of
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both the cpDNA and the mtDNA. These RFLPs are useful organelle markers in the cross.
b. We can see that all the progeny have inherited their mtDNA from the maternal parent R,
because all show the same R mtDNA fragment hybridized by the probe. This outcome is what
we might have predicted, on the basis of the predominantly maternal inheritance encountered
in this chapter. However, cpDNA is apparently inherited exclusively paternally, because all
progeny show the 5/4/1 pattern of the paternal plant S. This paternal inheritance is surprising,
but it is the only explanation of the data. In fact, all the gymnosperms studied so far show
paternal inheritance of cpDNA. The cause is unknown, but the phenomenon contrasts with
that in angiosperms.
c. From this cross, we can predict that all progeny will show the paternal 5/2/1 pattern for
cpDNA and the maternal 1.8-kb band for mtDNA.
Problems
1. How do the nuclear and organelle genomes cooperate at the protein level?
2. Name and describe two tests for organelle inheritance.
3. What is the basis for the green-white color variegation in the leaves of Mirabilis? If the
following cross is made,
what progeny types can be predicted? What about the reciprocal cross?
See answer
4. In Neurospora, the mutant stp exhibits erratic stop-start growth. The mutant site is known to
be in the mitochondrial DNA. If an stp strain is used as the female parent in a cross to a
normal strain acting as the male, what type of progeny can be expected? What about the
progeny from the reciprocal cross?
5. If a yeast cell carrying an antibiotic-resistance mutation in its mtDNA is crossed to a normal
cell and tetrads are produced, what ascus types can you expect with respect to resistance?
See answer
6. A new antibiotic mutation (antR) is discovered in a certain yeast. Cells of genotype antR are
mutagenized, and petite colonies are obtained. Some of these petites prove to have lost the
antR determinant.
a. What can you conclude about the location of the antR gene?
b. Why didn't all the petites lose the antR gene?
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7. Two corn plants are studied. One is resistant (R) and the other is susceptible (S) to a certain
pathogenic fungus. The following crosses are made, with the results shown:
What can you conclude about the location of the genetic determinants of R and S?
See answer
8. In Chlamydomonas, a certain probe picks up a restriction fragment length polymorphism in
cpDNA. There are two morphs, as follows:
If the following crosses are made,
what progeny types can be predicted from these crosses? Be sure to draw the DNA morphs
with their restriction sites. Also draw a sketch of the autoradiogram.
9. In yeast, the following cross is dihybrid for two mitochondrial antibiotic genes:
(MATa and MATα are the mating-type alleles in yeast.) What types of tetrads can be
predicted from this cross?
See answer
10. In the genus Antirrhinum, a yellowish leaf phenotype called prazinizans (pr) is inherited as
follows:
Explain these results on the basis of a hypothesis of cytoplasmic inheritance. (Explain both
the majority and the minority classes of progeny.)
11. You are studying a plant with tissue comprising both green and white sectors. You wish to
decide whether this phenomenon is due (1) to a chloroplast mutation of the type considered
in this chapter or (2) to a dominant nuclear mutation that inhibits chlorophyll production
and is present only in certain tissue layers of the plant as a mosaic. Outline the
experimental approach that you would use to resolve this problem.
See answer
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12. A dwarf variant of tomato appears in a research line. The dwarf is crossed as female to
normal plants, and all the F1 progeny are dwarfs. These F1 individuals are selfed, and all
the F2 progeny are normal. Each of the F2 individuals is selfed, and the resulting F3
generation is 3/4 normal and 1/4 dwarf. How can these results be explained?
13. Assume that diploid plant A has a cytoplasm genetically different from that of plant B. To
study nuclear–cytoplasmic relations, you wish to obtain a plant with the cytoplasm of plant
A and the nuclear genome predominantly of plant B. How would you go about producing
such a plant?
See answer
14. Two species of Epilobium (fireweed) are intercrossed reciprocally as follows:
The progeny from the first cross are backcrossed as females to E. hirsutum for 24
successive generations. At the end of this crossing program, all the progeny still are tall,
like the initial hybrids.
a. Interpret the reciprocal crosses.
b. Explain why the program of backcrosses was performed.
15. One form of male sterility in corn is maternally transmitted. Plants of a male-sterile line
crossed with normal pollen give male-sterile plants. In addition, some lines of corn are
known to carry a dominant nuclear restorer gene (Rf) that restores pollen fertility in
male-sterile lines.
a. Research shows that the introduction of restorer genes into male-sterile lines does not
alter or affect the maintenance of the cytoplasmic factors for male sterility. What kind of
research results would lead to such a conclusion?
b. A male-sterile plant is crossed with pollen from a plant homozygous for gene Rf. What
is the genotype of the F1? The phenotype?
c. The F1 plants from part b are used as females in a testcross with pollen from a normal
plant (rf/rf). What would be the result of this testcross? Give genotypes and phenotypes,
and designate the kind of cytoplasm.
d. The restorer gene already described can be called Rf-1. Another dominant restorer, Rf-2,
has been found. Rf-1 and Rf-2 are located on different chromosomes. Either or both of the
restorer alleles will give pollen fertility. With the use of a male-sterile plant as a tester,
what would be the result of a cross in which the male parent is:
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16. Treatment with streptomycin induces the formation of streptomycin-resistant mutant cells
in Chlamydomonas. In the course of subsequent mitotic divisions, some of the daughter
cells produced from some of these mutant cells show streptomycin sensitivity. Suggest a
possible explanation for this phenomenon.
See answer
17. In Aspergillus, a “red” mycelium arises in a haploid strain. You make a heterokaryon with
a nonred haploid that requires para-aminobenzoic acid (PABA). From this heterokaryon,
you obtain some PABA-requiring progeny cultures that are red, along with several other
phenotypes. What does this information tell you about the gene determining the red
phenotype?
See answer
18. Adrian Srb crossed two closely related species, Neurospora crassa and N. sitophila. In the
progeny of some of these crosses, a phenotype called aconidial (ac) appeared that lacks
conidia (asexual spores). The observed inheritance was:
a. What is the explanation for this result? Use symbols to explain all components of your
model.
b. From which parent(s) did the genetic determinants for the ac phenotype originate?
c. Why were neither of the parental types ac?
19. Several crosses between poky or nonpoky strains A, B, C, D, and E were made in
Neurospora. Explain the results of the following crosses, and assign genetic symbols for
each of the strains. (Note that poky strain D behaves just like poky strain A in all crosses.)
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20. The mtDNA of seven cytoplasmic petites was characterized by restriction-enzyme
analysis. The results showed that the mtDNA retained in each of these petites was as
indicated by the arcs in the diagram below.
Ten mit− mutants were fused with each of the petites to make heteroplasmons, and cells
from these heteroplasmon cultures were plated onto standard growth medium. Of the 70
combinations, some showed only petite phenotype colonies on the plate (represented by a
minus sign in the following grid), but the remainder showed some wild-type colonies as
well as petites (represented by a plus sign in the grid). These wild types must have arisen
by crossing-over between the petite and the mit point mutations. Use these results to locate
the approximate positions in the mtDNA of the genes that mutated to give the original mit−
cultures.
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Unpacking the Problem
1. What is mtDNA?
2. Draw an mtDNA molecule, showing at least five specific genes found in mtDNA.
3. What is a cytoplasmic petite?
4. What is the nature of a cytoplasmic petite at the mtDNA level?
5. Why is it appropriate to represent petite DNA by an arc of a circle?
6. Briefly describe a restriction-enzyme analysis that might have been used to determine
the extent of the DNA retained by a petite.
7. What is a mit− mutant? How does a mit− mutant compare with a petite mutant? Sketch a
mit−, a petite, and a normal colony.
8. Are mit− mutants drug resistant?
9. Give another word for a heteroplasmon or make up a description term for the concept
yourself.
10. How would you make the heteroplasmons in this experiment? Would auxotrophic
markers be useful?
11. In what sense is the word fused used in this problem?
12. What happens to yeast cells on growth medium?
13. Draw a typical plate representing a + result in the grid.
14. Draw a typical plate representing a − result in the grid.
15. What exactly do the + and − results mean? Do they refer to complementation or
recombination?
16. Are all the mit− mutants different in their behavior?
17. Do all the petites show different behavior in combination with the mit− mutants?
18. If some mit− mutants show the same behavior, how is this possible if the petites are
different according to the restriction analysis?
21. In yeast, an antibiotic-resistant haploid strain antR arises spontaneously. It is combined
with a normal antS strain of opposite mating type to form a diploid culture that is then
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allowed to go through meiosis. Three tetrads are isolated:
a. Interpret these results.
b. Explain the origin of each ascus.
c. If an antR grande strain were used to generate petites, would you expect some of the
petites to be antS? Explain your answer.
See answer
22. In yeast, two haploid strains are obtained that are both defective in their cytochromes; the
mutants are designated cyt1 and cyt2. The following crosses are made:
One tetrad is isolated from each cross:
a. From these tetrad patterns, explain the differences in the two underlying mutations.
b. What other ascus types could be expected from each cross?
c. How might the two genes interact at the functional level?
23. The mtDNAs from two haploid strains of baker's yeast are compared. Strain 1
(mating-type α) is from North America, and strain 2 (mating-type a) is from Europe. A
single restriction enzyme is used to fragment the DNAs, and the fragments are separated
on an electrophoretic gel. The sample from strain 1 produces two bands, corresponding to
one very large and one very small fragment. Strain 2 also produces two bands, but they are
of more intermediate sizes. If a standard diploid budding analysis is performed, what
results do you expect to observe in the resulting cells and in the tetrads derived from them?
In other words, what kinds of restriction fragment patterns do you expect?
See answer
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24. In yeast, some strains are found to have in their cytoplasm circular DNA plasmids that are
2 micrometers (μm) in circumference. In some of these strains, this 2-μm DNA has a single
EcoRI restriction site; in other strains, there are two such sites. A strain with one site is
mated to a strain with two sites. All the resulting diploid buds are found to contain both
kinds of 2-μm DNA.
a. Is the 2-μm DNA inherited in the same fashion as mtDNA?
b. If you used radioactive 2-μm DNA as a probe, predict the results of hybridizing it to a
Southern blot of EcoRI-treated DNA of ascospores from these diploid cells.
See answer
25. Circular mitochondrial DNA is cut with two restriction enzymes, A and B, with the
following results:
(The arrows indicate the bands that bound a radioactive mt rRNA-derived cDNA probe in
a Southern blot.) Draw a rough map of the positions of the restriction site(s) of A and B,
and show approximately where the mt rRNA gene is located.
26. You are interested in the mitochondrial genome of a fungal species for which genetic
analysis is very difficult but from which mtDNA can be extracted easily. How would you
go about finding the positions of the major mitochondrially encoded genes in this species?
(Assume some evolutionary conservation for such genes.)
See answer
27. a. Early in the development of a plant, a mutation in cpDNA removes a specific BgIII
restriction site (B) as follows:
In this species, cpDNA is inherited maternally. Seeds from the plant are grown, and the
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resulting progeny plants are sampled for cpDNA. The cpDNAs are cut with BgIII, and
Southern blots are hybridized with the probe P shown. The autoradiograms show three
patterns of hybridization:
Explain the production of these three seed types.
b. In Gryllus (a cricket) and Drosophila, rare females have been found that show mixtures
of two mtDNA types, differing by the presence or absence of one specific restriction site.
In the progeny of these females, each and every individual also proves to be a mixture of
the parental mtDNA types. Contrast these results with the results from part a and with
other results in this chapter.
28. The mitochondrial genome of the turnip is a large, circular molecule 218 kb in size with a
pair of 2-kb direct repeats located about 83 kb apart. However, when turnip mtDNA is
examined carefully, three molecular types are seen: the 218-kb circle just described, a
135-kb circle bearing a single 2-kb repeat, and an 83-kb circle also bearing only one copy
of the 2-kb repeat. Propose a model to explain the presence of the two smaller molecular
types.
29. Reciprocal crosses and selfs were performed between the two moss species Funaria
mediterranea and F. hygrometrica. The appearance of the sporophytes and the leaves of
the gametophytes are shown in the accompanying diagram. The crosses are written with
the female parent first.图片已删
a. Describe the results presented, summarizing the main findings.
b. Propose an explanation for the results.
c. Show how you would test your explanation; be sure to show how it could be
distinguished from other explanations. 图片已删
(Diagrams after C. H. Waddington, An Introduction to Modern Genetics, Macmillan,
1939.)
30. The pedigree below shows a very unusual inheritance pattern that actually did exist. All
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progeny are shown, but the fathers in each mating have been omitted to draw attention to
the remarkable pattern.
a. State concisely exactly what is unusual about this pedigree.
b. Can the pattern be explained by 图片已删 Explain.
31. Consider the following pedigree for a rare human muscle disease:
a. What is the unusual feature that distinguishes this pedigree from those studied earlier in
this book?
b. Where in the cell do you think the mutant DNA resides that is responsible for this
phenotype?
32. The following pedigree shows the recurrence of a rare neurological disease (large black
symbols) and spontaneous fetal abortion (small black symbols) in one family. (Slashes
mean that the individual is deceased.) Provide an explanation for this pedigree in regard to
cytoplasmic segregation of defective mitochondria.
Chapter 21*
3. Maternal inheritance of chloroplasts results in the green-white color variegation observed in
Mirabilis.
5. Both yeast parents contribute mitochondria to the cytoplasm of the resulting diploid cell.
Subsequent meiosis shows uniparental inheritance for mitochondria. Therefore, 4:0 and 0:4
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asci will be seen.
7. The genetic determinants of R and S are cytoplasmic and are showing maternal inheritance.
9. Both yeast parents contribute mitochondria to the cytoplasm of the resulting diploid cell.
Recombination of mtDNA can take place in these heteroplasmons, but cytoplasmic
segregation results in tetrads that contain either one of the parental mtDNA genotypes or
one of the reciprocal recombinant types.
11. If the mutation is in the chloroplast, reciprocal crosses will give different results; whereas,
if it is in the nucleus and dominant, reciprocal crosses will give the same results.
13. After the initial hybridization, a series of backcrosses using pollen from plant B will result
in the desired combination of cytoplasm A and nucleus B. With each cross, the female
contributes all of the cytoplasm and half the nuclear contents, whereas the male contributes
half the nuclear contents.
16. The suggestion is that the mutants are heteroplasmons and contain both wild-type (sm-s)
and mutant (sm-r) organelle DNA. Subsequent mitotic divisions can lead to cytoplasmic
segregation such that daughter cells contain only one organelle DNA type.
17. The red phenotype in the heterokaryon indicates that the red phenotype is caused by a
cytoplasmic organelle allele.
21. a. and b. Each meiosis shows uniparental inheritance, suggesting cytoplasmic inheritance.
c. Because antR is probably mitochondrial and because petites have been shown to result
from deletions in the mitochondrial genome, antR may be lost in some petites.
23. Some tetrads will show strain-1 type, some will show strain-2 type, and some will be
recombinant.
24. a. No; in diploid budding, all the progeny receive one type of mtDNA. Most likely it is a
plasmid or episome.
b. Three bands, one at 2μ, the others totaling 2μ.
26. First, prepare a restriction map of the mtDNA by using various restriction enzymes.
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Assume evolutionary conservation and, on a Southern blot, hybridize equivalent fragments
from yeast or another organism in which the genes have already been identified.
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