Journ. of Genetics 48: 80-98. 1947
HETEROCHROMATIZATION AS A CHANGE OF CHROMOSOME CYCLE
By A. A. PROKOFYEVA-BELGOVSKAYA
Institute of Cytology, Histology and Embryology of the Academy of Sciences of the U.S.S.R.

(With Nine Text-figures)

A survey of the problem of heterochromatization shows that we must take into account two classes of facts. On the one hand we have facts showing that a chromosome is made up of inert (heterochromatic) and active (euchromatic) regions, with different properties (Henking, 1891; Wilson, 1905-12; Heitz, 1928, 1929, 1933a,b, 1934; Muller & Painter 1932; Darlington & La Cour, 1940, 1941; Caspersson, 1940a b, 1941; White, 1940). On the other hand, facts have accumulated which show that the process of heterochromatization (heteropycnosis) is reversible (Mohr, 1915; McNabb, 1928; Junker, 1923; Shinke, 1937; Prokofyeva-Belgovskaya, 1937a, 1939c; Darlington & La Cour, 1940; Dobzhansky, 1944).

The work here described is intended to answer four questions:

  1. Is the conception that the chromosomes are built up of euchromatic 'euchromatin' and heterochromatic 'heterochromatin' correct?
  2. What are the causes and conditions of heterochromatization?
  3. What is the role of heterochromatization in character development?
  4. What is the nature of heterochromatization?

STRUCTURE AND PROPERTIES OF EUCHROMATIC AND HETEROCHROMATIC REGIONS

It is now definitely established that the euchromatic and heterochromatic regions do differ significantly in their fine morphological structure. The chromosome is throughout built up of chromonemata and chromioles, these two constituents being basic both euchromatic and heterochromatic regions.

The structure of the proximal, distal and interstitial heterochromatic regions is similar to that of the rest of the chromosome (Prokofyeva-Belgovskaya, 1937a,b,c, 1938, 1939a,c; Tiniakov, 1936; Frolowa, 1936a,b,c; Bauer, 1936). But in spite of the likeness of the basic morphological elements throughout the chromosome, the chromonemata and chromioles of the heterochromatic regions have a series of characteristic properties different from those of the euchromatic regions. These properties presumably depend on their biochemical composition, and particularly, according to Caspersson (1940a,b, 1941), on the structure of their proteins.

The characteristic, cytologically detectable properties of heterochromatic (inert) regions are as follows:

  1. Overcharging with thymonucleic acid (Caspersson, 1940; Darlington & La Cour, 1940).
  2. The capacity of all heterochromatic parts to conjugate with one another (McClintock, 1933; Prokofyeva-Belgovskaya, 1937b,c, 1938, 1939a; Bauer, 1936; Hinton, 1945).
  3. Mechanical weakness of their chromonemata, increasing the frequency of chromosomal rearrangement and crossing-over in them (Prokofyeva-Belgovskaya & Khvostova, 1939; Kaufmann, 1939; Prokofyeva-Belgovskaya & Belgovsky, 1943).
  4. A strong tendency to heterochromatization (heteropycnosis) (Heitz, 1928, 1929, 1933a,b).

THE CYTOLOGICAL PICTURE OF HETEROCHROMATIZATION

Heterochromatization or heteropycnosis is a state of a chromosome section when

  1. It contains an increased amount of thymonucleic acid.
  2. Its chromonema is often twisted into a tight spiral.
  3. During most of the cell cycle it has, in distinction from other chromosomal sections, the form of a deeply staining metaphasic chromosome body. Such a form has frequently been described in different animal and plant tissues.

Heterochromatization is a condition characteristic of the mitotic stage. But whole chromosomes or definite (heterochromatic or inert) chromosome parts may assume this condition long before the nucleus as a whole enters the mitotic stage.

Fig. 1. Heterochromatization of the inert region and the active region situated near the inert one.

In Drosophila. Heitz (1933a,b, 1934) was the first to study heterochromatization. The heterochromatized sections (X, Y, and the proximal parts of II and III) have the form of typical metaphasic chromosome bodies in the prophase of larval ganglion cells. In the nuclei of salivary gland cells the heterochromatization is expressed in another way, on account of the peculiar structure of chromosomes in these cells. In comparison with the euchromatic regions where all chromomeres and chromonemata conjugate intimately with one another, the chromomeres forming bands and the chromonemata the unstaining space between them, conjugation of chromonemata is much reduced in the proximal inert regions. In inert regions, instead of bands, we see single chromomeres which sometimes contain considerable quantities of thymonucleic acid. This acid is also found in chromonemata.

One of the most typical peculiarities of the 'mitotic' condition of a chromosome is the separation or repulsion of the chromatids, connected with an increased content of thymonucleic acid. This is the basis of the cytological condition of heterochromatic regions in the salivary glands. In other words, heterochromatization in salivary glands is a shift of the cycle of certain chromosomal regions towards mitosis (Fig. 1).

EXPERIMENTAL PART

A. The interaction of heterochromatic and euchromatic regions: the reversibility of heterochromatization

Is it true that heterochromatic sections always have a heterochromatic appearance, while euchromatic ones have a euchromatic appearance, or, in other words, that a chromosome is built of two substances, euchromatin and heterochromatin? To answer this question I studied the influence of active regions on the heterochromatic structure of in sections, and that of inert regions on the euchromatic structure of active sections.

Fig. 2. The behaviour of the long inert region inserted into the active one. The origin of a deficiency. a, upper view; b, side view. The connexion of inserted inert region with a main chromosome body is very weak wm4.

(1) Change of the state of inert regions under the influence of active ones

I investigated sc8, wm4, wm5, and rst3 stocks, in all of which a rearrangement has cause the inclusion of a part of the inert region (normally heterochromatic) in the euchromatic region. Under these conditions the heterochromatization of inert regions decreased greatly the region inserted sometimes assuming an almost euchromatic condition, and becoming indistinguishable from the neighbouring active section. Thymonucleic acid disappears from the chromonemata, which conjugate more intimately and become invisible, while the homologous chromomeres form typical disks (Fig. 2a). The smaller is the inert region inserted, the stronger is the influence of active sections upon it, as is seen on comparing the conditions of the inserted sections in the sc8, wm4 and rst3 chromosomes. This is apparently one of the causes of the euchromatic appearance of all short interstitial inert regions. When long inert regions are inserted into active ones, as in rst3 and wm4, their terminal portions often tend to conjugate, forming a small ring chromosome which is shed by the main chromosome body (Fig. 2b) (cf. McClintock, 1938).

(2) Change of state of active regions under the influence of inert ones

Inert regions have the opposite influence on active ones in their neighbourhood. I investigated a series of stocks (sc8, wm5, rst3, wm4) in which a rearrangement has brought an active region near to the inert one. In all cases the heterochromatization of the inert region spread to the neighbouring active sections, which took on a typical heterochromatic condition. Their chromonemata conjugated less intimately, and the bands assumed the form of separate chromomeres. The smaller is the active section brought close to the inert region, and the longer the latter, the stronger is the heterochromatization of the active region. The active section is sometimes so highly heterochromatized that it becomes quite indistinguishable from the neighbouring inert region (Prokofyeva-Belgovskaya, 1937a, b, 1939c).

These observations definitely show that the processes leading to heterochromatization or euchromatization of chromosome sections are reversible. The heterochromatic condition is not a property of heterochromatic or inert regions only, nor is the euchromatic condition confined to euchromatic or active regions. These two conditions are cyclical physiological states of a chromosomal section, and under appropriate conditions any chromosomal section may show either of the two structures.

B. Conditions of heterochromatization

In order to find out under what conditions heterochromatization occurs, I investigated the influences of:

  1. The neighbourhood of the centromere.
  2. The presence of an additional Y-chromosome.
  3. Sex.
  4. The direction of the cross.
  5. The temperature.
  6. The age of the parents.

As the states of the inert and the neighbouring active regions change similarly, we shall consider the phenomenon of heterochromatization irrespective of whether it occurs in the inert region, in the active one, or in both at once. The interdependence of the states of active and adjacent inert regions is so intimate that they must often be considered as a single region for this purpose.

(1) The percentage of heterochromatization

One phenomenon was always seen when the degree of heterochromatization of a particular chromosomal section was studied in different stocks and under different conditions. A section which is in the euchromatic state has the same structure in all the salivary gland nuclei of a given larva, whilst if it is in the heterochromatic state it varies considerably in different salivary nuclei of one larva.

When a section is in the heterochromatic condition in some nuclei one can hardly find two nuclei in which it has the same degree of heterochromatization. Even two neighbouring nuclei may differ very sharply, the same section being euchromatic in one and completely heterochromatic in the other (Fig. 3).

It was therefore necessary to find an index which would adequately represent the state of the nuclei in a given stock. For this purpose we chose the 'percentage of heterochromatization', which was determined as follows. I selected a sufficient number of good slides, each of which bore a pair of salivary glands from a larva of a given stock. On each slide ten nuclei in which the section investigated was in a position convenient for cytological analysis were selected under a low magnification (Apo. 10; K. 10x). This magnification allowed the marking of nuclei suitable for investigation, but was quite insufficient to determine the state of the chromosome section; so the choice of ten nuclei per slide was entirely random in this respect. The state of the section under investigation was determined under a high magnification (Apo. 90, 1.4; K. 15x). The percentage of heterochromatization is the percentage of nuclei in which the given section was in the heterochromatic condition. Numerous observations have shown that this percentage has a constant value characteristic of a given stock under definite conditions.

Fig. 3. The possible states of the 1AB1-20ABC region of the sc8 chromosomes.
a, euchromatic state; b, c, heterochromatic state.

Table 1. State of the 1AB1 (y-ac) 20ABC region in the sc8 chromosome, T and U larvae

  Genotype No. of nuclei
investigated
No. and percentage of nuclei
with euchromatic region
No. and percentage of nuclei
with heterochromatic region
No. and percentage of nuclei
with hetero-euchromatic region
T
sc8
sc8
200 69, 34.5 77, 38.5 54, 27
U
sc8
Y
200 168, 84 32,16 0

(2) Influence of sex on the state of the chromosomal section

This investigation was carried out on the X-chromosome of the sc8 stock, as were most of the others. I studied the inert region 20ABC and the active section 1AB1 immediately adjacent to it, which includes the genes y and ac. The distal end of this chromosome is obviously different in the two sexes (Table 1). In males both the regions are in a well-expressed euchromatic condition. It is often impossible to see where the active region ends and the inert one begins. This section is very rarely heterochromatized in males, and if so the degree of its heterochromatization is very low (Fig. 4).

In females the 20ABC and 1AB1 sections are highly heterochromatic in many nuclei, the degree of heterochromatization being higher in the inert region and gradually decreasing towards the distal end. The degree varies from nucleus to nucleus, and one can observe both the euchromatic and heterochromatic condition of the whole section in question, and all intermediate conditions.

The percentage of heterochromatization of the y-ac section is 16% in sc8 males, and 38.5% in sc8 females. In several cases (27%) I observed differences in the structure of homologous chromosomes in the most sensitive inert region (20ABC) and in the adjoining active region (1AB1): in the chromosome derived from the father these regions had been heterochromatized, while in that derived from the mother they were in an euchromatic condition (Fig. 9). The nature of this phenomenon will be described later.

Fig. 4. Heterochromatization of the 1AB1-20ABC region of the sc8 chromosome in males (a, b) and females (c).

(3) Influence of an additional Y-chromosome on the state of the chromosome section

I studied the structure of the 1AB1-20ABC section containing the y and ac loci of sc8 females with an additional short arm of the Y-chromosome. The highly heterochromatic condition of the 20ABC and 1AB1 sections, occurring in many nuclei of ordinary sc8 females, was strongly, suppressed in this stock (Fig. 5). In the presence of an additional Y-chromosome these sections become more euchromatic, and the whole picture of their structure is like that observed in sc8 males (Table 2).

(4) Influence of the direction of the cross on the state of the chromosome section

I studied the state of the 1AB1-20ABC section in the sc8 chromosome of heterozygous sc8 x y ac v females obtained in two reciprocal crosses: sc8 x y ac v, and y ac v x sc8

(Figs. 6, 7). The first group of F1 females received the sc8 chromosome from their mothers, the second group from their fathers. The degree of heterochromatization of the 1AB1 section of this chromosome differed with the direction of the cross. In the first group its percentage of heterochromatization was 20, in the second 71, or about 3.5 times as much (Table 3).

Fig. 5. The 1AB1-20ABC region of the sc8 chromosome in females with an additional Y-chromosome.

Table 2. Influence of an additional Y-chromosome on the state of the 1AB1 (y-ac) 20AB
region in the sc
8 chromosome, female larvae

Origin No. of nuclei
investigated
No. and percentage of nuclei
with euchromatic region
No. and percentage of nuclei
with heterochromatic region
sc8 female x XY* male 200 174, 87 26, 13
sc8 female x y ac v male 200 160, 80 40, 20

*y ac v f chromosome attached to the short arm of Y.

 

Fig. 6. The 1AB1-20ABC region of the sc8 chromosome in T sc8 x U y ac v, female larvae.

(5) Influence of the centromere on the state of the chromosome section

I investigated this influence on wm4, wm5, mMed and rst3 stocks of D. melanogaster (Fig. 8). These stocks carry chromosomal rearrangements in which one break has occurred at the centromere of chromosome X or IV, the other at the w locus; so the active region 3C2 is very close to the centromere. The euchromatic state of this section is slightly disturbed in these conditions. The degree of heterochromatization occurring in the region under the direct influence of the centromere is very low, but, nevertheless, quite definite. These observations (Table 4) suggest that the centromere is one of the intracellular factors which may cause heterochromatization of a chromosome section.

Fig. 7. The 1AB1-20ABC region of the sc8 chromosome in ♀ y ac v x ♂ sc8, female larvae.

Table 3. Influence of the direction of the cross on the 1AB1 (y -ac) 20 ABC region in the sc8 chromosome, ♀ larvae

Origin No. of nuclei
investigated
No. and percentage of nuclei
with euchromatic region
No. and percentage of nuclei
with heterochromatic region
Percentage
of mosaics
sc8 ♀ x y ac v 200 160, 80 40, 20 439
y ac v ♀ x sc8 200 58, 29 142, 71 21.92

 

Fig. 8. a, b. The 3C section situated near the inert region. (a) wm4; (b) wm5;
(c, d) the 3C section situated in the immediate neighbourhood of the centromere; (c) wm4, (d) rst3.

(6) Influence of temperature on the state of the chromosome section

The laws according to which temperature influences chromosomal regions are still from clear. The action of temperature is probably different at different stages of development. I investigated heterozygous female larvae from the cross y sc vsc8 x ♂. The egg were laid at 25° C., and 6 hr. later portions of the cultures were transferred to 14 and 30° C.

Table 4. Heterochromatization and mosaicism in the wm4 and wm5 lines.
Influence of the centromere on the state of the
3C2 (white) region, larvae

Stock No. of nuclei investigated No. and percentage of nuclei
with euchromatic region
No. and percentage of nuclei
with heterochromatic region
Expression of character in adult fly
wm4 150 110, 73.33 40, 26.67 Dark mottled
wm5 160 30, 18.75 130, 81.25 Light mottled

 

Table 5. Influence of temperature on the state of the 1AB1 (y-ac) 20 ABC region
in the sc8 chromosome, larvae

 

Temp. C. No. of nuclei investigated No. and percentage of nuclei
with euchromatic region
No. and percentage of nuclei
with heterochromatic region
14° 160 107, 66.9 53, 33.1
25° 200 58, 29 142, 71
30° 130 82, 63.1 48, 36.9

Table 6. Influence of parental age on the state of the 1AB1 (y-ac) 20ABC region
in the sc
8 chromosome of larvae from y ac v x sc8

  Exp. 1 Exp. 2 Exp. 3
Age of parents
in days
Euchromatic Heterochromatic Euchromatic Heterochromatic Euchromatic Heterochromatic
% % % % % %
1-5 33 67 42 58 31 69
5-10 22 78 36.5 63.5 20 80
10-15 21 79 25 75 20 80
15-20 17 83 21 79 20 80
20-25 18 82 21 79 - -
25-30 18 82 - - - -

The results are shown in Table 5. Under standard conditions (25° C.) the percentage of heterochromatization of the 1AB 1-20ABC region of the sc8 chromosome of this stock is 71. In larvae transferred to 14° C. after 6 hr. it is 33. Thus heterochromatization is suppressed when development proceeds at the low temperature. The high-temperature experiment gave very similar results. The percentage of heterochromatization in larvae transferred to 30° C. after 6 hr. was 37, and was thus again decreased.

(7) The influence of parental age on the state of the chromosome section

I investigated the F1 larvae from y ac v x sc8 ♂; the parents were transferred to fresh food every fifth day. Three experiments lasted 30, 23 and 20 days respectively. The y-ac region of the sc8 chromosome in larval salivary glands was examined. The results are presented in Table 6.

The condition varies with the age of the parents. Their ageing results in a progressive heterochromatization of the region 20ABC-1AB1 in their progeny. There is reason to believe that ageing causes a progressive heterochromatization of the nuclei of the parents, and that this process affects the condition of the most sensitive chromosome regions in the progeny. The dependence of these regions' state in the progeny upon their condition in parents is also evidenced by the influence of the direction of the cross, and has also been shown by Belgovsky and the author (unpublished) in their study of the relation between the frequency of minute rearrangements and the degree of heterochromatization of a chromosome region.

The number of nuclei studied was 100 in each case, except in Exp. I (25-30 days), Exp. I (5-10 days), Exp. II (25-30 days) and Exp. III (15-20 days), in which 50, 88, 90 and 70 nuclei respectively were observed.

C. Significance of heterochromatization for character development

A transfer of active sections to the neighbourhood of inert regions regularly leads to the heterochromatization of these regions. No other cytological changes were ever observed in these cases.

After the publication of Schultz's data (Morgan, Bridges & Schultz, 1936-8) I carried out a new and extensive investigation of stocks mosaic for white (wm4, wm5, wmMed), i.e. of stocks similar to those used by Schultz (Prokofyeva-Belgovskaya, 1939b,c). This investigation fully substantiated my first observations. No losses of active chromosome sections adjacent to inert regions were observed. The chief change is the regular assumption of the heterochromatic condition by such sections. The 3C2 section, containing the w locus in its right part, could be identified in the heterochromatic condition in every nucleus of all stocks investigated, although according to Schultz it was missing in his mosaic strains. Schultz was right only in the sense that in his cases the 3C2 section frequently could not be observed as an euchromatic structure, but it remained in the nucleus in the heterochromatic state. The nuclei in the stocks investigated proved very variable, some having the 3C2 region in the euchromatic condition. some in the heterochromatic, and others in different intermediate states.

Heitz (1928, 1929, 1932, 1933a b, 1934) showed that heterochromatization of any chromosome section shortens its metabolic stage, since it passes through the whole cell cycle as a compact metaphasic body. This suggests that the greater the number of nuclei in which a chromosome section influencing the development of a character is heterochromatic, the more will development occur as if this section had been lost, i.e. the more strongly will a recessive character be expressed in a mosaic strain. To test this hypothesis I compared my cytological data on heterochromatization of certain chromosome sections with data on mosaicism in the same stocks obtained by other workers independently.

The degree of mosaicism for eye colour differs in the wm4 and wm5 stocks. In wm4 colourless ommatidia are scattered on a coloured background, so most of the eye surface shows the dominant character. In wm5 the recessive character prevails, the background is colourless, the pigmented ommatidia form coloured spots. In the salivary glands of wm4 larvae the 3C2 section, which plays an active part in forming pigment in ommatidia, Malphighian tubes and testes, is almost adjacent to the centromere, and is in a slightly heterochromatic state in 26.7% of the nuclei. In the wm5 stock this section is near to the main bulk of the proximal inert region of chromosome IV, and in 81.25% of nuclei is in a well-expressed heterochromatic state (Table 4). In other words, increasing heterochromatization of this active section shifts the character towards the recessive manifestation.

* The numerous other cases of alterations in the normal activity of euchromatic regions approximated to the heterochromatic ones (Dubinin, 1936; Belgovsky, 1938, 1944; Demerec, 1940,1941; Demerec & Slizynska, 1937; Kaufmann, 1942; Schultz & Caspersson, 1939; Sidorov, 1936 and others) are presumably to be explained also in terms of a change of their cycles. The inactivation of the dominant K gene occurring in the micronucleus of Paramecium aurelia (Sonneborn, 1946) must also be dependent upon the shift of the micronucleus chromosomes towards the mitotic condition.

This hypothesis was further tested by comparing my cytological data on heterochromatization of the 1AB1 section, containing the y and ac loci in the sc8 chromosome the genetic data of Noujdin (1944) on mosaicism in the same strain (Table 3). These show that mosaicism is intimately connected with a change of the physiological state of the active chromosome section, namely, its conversion from the euchromatic to heterochromatic state. This conversion depends on the approximation of this section to the inert region, which causes it to react to different genetical, developmental, environmental conditions by changing its cycle.*

D. Heterochromatization and crossing-over

We saw that heterochromatization is chiefly expressed by an increased thymonucleic acid content of the chromosomes and a weakening of the conjugational properties at chromonemata. These do not conjugate with one another when in the heterochromatic state. These observations led to the suggestion that heterochromatization and euchromatization on the one hand, and the conjugation of chromosomes in meiosis on the other, depend on common causes, heterochromatization and the suppression of meiotic conjugation being phenomena of the same kind.

To check this hypothesis I compared the influence of the following factors on these two processes: temperature, inert regions, rearrangements which insert inert sections into an active region, the position of the centromere, and the parental age. I had for comparison my own cytological data on the one hand and extensive genetical data on crossing-over on the other.

(1) Influence of temperature

The development of young sc8 larvae at 14 and 30° C. converts the heterochromatic state of section 1AB1-20ABC into the euchromatic one (see § B). The thymonucleic acid content of the chromonemata decreases, while their conjugational properties increase. These observations agree completely with Plough's (1917, 1921) and Mather's (1939) data on the effect of temperature on crossing-over. A decrease of temperature from 25 to 13°C. and an increase to 30° C. increases the percentage of crossing-over, which is maximal at 13 and 30° C. Plough showed that the sensitive stage at which temperature changes are effective in altering crossing-over values is that of the early oocytes, i.e. the stage at which conjugation occurs. White's (1934) studies on the influence of temperature on chiasma frequency led to similar results. Mather's more recent work fully substantiated Plough's, and also revealed some important new facts which we shall consider later.

(2) The influence of inert regions

The inert regions, mostly confined to the proximal ends of chromosomes, near the centromeres, persist in the heterochromatic state during most of the cell cycle. The active regions located near the inert ones are also in the heterochromatic condition (see §§ A, B). Thus inert regions which are heterochromatic induce heterochromatism in neighbouring active sections.

The influence of inert regions on crossing-over has been studied thoroughly. Crossing-over is strongly suppressed in the proximal ends of all chromosomes (Kikkawa, 1932; Offermann & Muller, 1932; Beadle, 1932), which is presumably due to a strong decrease in the conjugation properties of these regions.

(3) The influence of chromosomal rearrangements involving the insertion of an inert section into the active region

As shown in § A, when an inert region is inserted into an active one (wm4, rst3) its heterochromatization decreases considerably. Complete euchromatization was seen comparatively rarely, but heterochromatization of region 20 of the X-chromosome in wm4 and rst3was suppressed in almost all nuclei. The degree of heterochromatization of the inert section inserted depends largely on its size. Mather (1939) showed that the percentage of crossing-over in inserted inert sections is much increased. His data on crossing-over in the inert region 20 in rst3 and sc8 fully agree with our data on the heterochromatization of the same section (see also Offermann, Stone & Muller, 1931; Offermann & Muller, 1932; Beadle, 1932).

(4) The influence of the centromere

I showed (§B (5)) that the centromere is an agent of heterochromatization, though a less effective one than the inert regions. When the active region 3C 2 in the wm4 line is transferred nearer to the centromere, it undergoes heterochromatization, though not so marked as when it adjoins the inert region of chromosome IV in the wm5 line. The degree of heterochrornatization varies much less between nuclei in line wm4 than in wm5, where the sensitive inert region, responding to slight physiological differences between cells, changes the condition of the adjacent 3C2 region containing the w locus, thus considerably increasing its variability.

Mather's results (see also Graubard, 1932) on the influence of the inert regions and of the centromere on crossing-over fully agree with these observations. He concluded that, though crossing-over reduction is largely brought about by the centromere, it is not the latter which is responsible for the variation of the crossing-over percentage with temperature, but the influence of the inert regions sensitive to the changing conditions. In the sc8 and the rst3 lines the percentage of crossing-over is more affected by temperature in the chromosomal regions into which the inert region has been inserted by inversion than at the centromere. These regularities appear to be the same as those observed by me of the effect of the centromere and inert regions on heterochromatization, i.e. the centromere causes heterochromatization (though a slight one), while variation of heterochromatization between nuclei is related to inert regions which show a specific response to the Physiological peculiarities of the cells containing them.

(5) The influence of parental age

It was found (§B) that the ageing of the parents causes a progressive heterochromatization in their progeny of the most sensitive inert regions and of the active ones in their vicinity. There is reason to suppose that ageing causes a progressive heterochromatization of the cell nuclei in the parents, and that this process tells upon the condition of the most sensitive regions in their progeny. A comparison of this evidence with that on the influence of age on crossing-over (Bridges, 1927, 1929) leads to the belief that the two phenomena are parallel. Ageing is accompanied by a reduction of the capacity of the chromosomes for conjugation and by a drop in the percentage of crossing-over.

DISCUSSION

(1) Heterochromatization as a change of chromosome cycle

Considering my data on heterochromatization in connexion with our general knowledge of chromosome behaviour I conclude that heterochromatization is intimately connected with the regular chromosome changes during the cell cycle. The differences in the absorption curves of the discoidal and heterochromatic (chromocentral and interstitial) chromosome regions described by Caspersson (1940a, b, 1941) seem to be due to differences in the cycles of these regions. Heterochromatization of the inert regions and the active ones which are located in their vicinity is identical with those changes in the chromosome properties that ordinarily attend the transition of the nucleus from the metabolic stage to division. The heterochromatic chromosome sections (active or inert) are characterized by a considerable acceleration of prophasic processes and a considerable retardation of telophasic ones, leading to a great shortening of the metabolic stage of these chromosome regions. Heterochromatization is a change of chromosome cycle.

However, the conditions which may lead to this result vary with the chromosome region. If a minute disturbance in the biochemical conditions of the cell is enough to shift the cycle of the inert region in either direction, it seems to be more difficult to shift the cycle of the active chromosomal regions in the metabolic nucleus. In the cases which I studied it was only the inert regions, and those among the active ones which were brought near to then by rearrangement, which reacted visibly to changes in intracellular conditions brought about by temperature, by an extra Y-chromosome, by chromosomal rearrangement, by the direction of the cross, or by the age of the parents.

For the organism as a whole shifts of the chromosome cycle during the metabolic stage are of particular importance. Those chromosome regions which remain in the condition of heterochromatic mitotic bodies in metabolic nuclei will be 'inert'. Their activity and the role they play in developmental processes will thus be reduced; and if they are strongly heterochromatized they will probably be entirely eliminated from these processes as specific chromosome regions. When tissues and organs are being developed from cells containing them, an effect of physiological 'loss' of these regions will be observed, and characters determined by them will tend to show a recessive expression. The hypothesis suggested by Schultz, who explains mosaicism as a result of chromosomal deficiency for an active region, has some physiological grounds.

(2) Genetical and environmental conditions of the chromosomal cycle

A study of the heterochromatization of the y-ac section of the sc8 chromosome in salivary gland nuclei of individuals of constitutions showed different percentages of heterochromatization, constant in each category (Table 7) and increasing in the order given. On analysing these results as to the cycle of the chromosome we find that the duration of the metabolic stage of this section is shortest in y ac v/sc8 females, and longest in sc8/XY females. This series corresponds strictly with the types of mosaicism found in these categories of flies by Noujdin (1944). We are thus led to the conclusion that the hereditarily conditioned degree of mosaicism of a given category of flies is due to the hereditary character of the cycle of their y-ac section.

sc8 sc8 sc8 sc8 and y ac v
XY* Y y ac v sc8 sc8
* y ac v f chromosome attached to the short arm of the Y.

Table 7. Hereditary determination of the chromosome cycle of the 1AB1 (y-ac) 20ABC region in sc8 chromosome

Origin Genotype No. of nuclei
investigated
Euchromatic
%
Heterochromatic
%
Hetero-Euchromatic
%
sc8 x XY sc8/XY* 200 87 13 0
sc8 x sc8 sc8/Y 200 84 16 0
sc8 x y ac v sc8/y ac v 200 80 20 0
sc8 x sc8 sc8/sc8 200 34 39 27
y ac v x sc8 y ac v/sc8 200 29 71 0

* y ac v f chromosome attached to the short arm of the Y.


Fig. 9. The 1AB1-20ABC region of the sc8 chromosome in female larvae.

If we consider the facts in the light of the interpretation given to the cycle of chromosomal regions, we reach the following result: It is not irrelevant to the cycle of a given chromosomal region whether it was in a heterozygous or homozygous condition during the meiosis of the parents. A y-ac section which underwent meiosis in a homozygous state (in a sc8 chromosome derived from the mother in the cross sc8 x y ac v) has a longer metabolic stage in the progeny. A y-ac which underwent meiosis in a heterozygous state, being in a heteropyknotic condition (in a sc8 chromosome derived from the father in the cross y ac v x sc8 ) has a shorter metabolic stage in the progeny. In the chromosome derived from the father the y-ac region has been heterochromatized, in that derived from the mother it is in the euchromatic condition (Fig. 9). This cytological evidence goes to confirm the correctness of Noujdin's (1944) conclusion as to the genetic heterogeneity of chromosomes of homozygous females in the pure line sc8.

We are thus led to the conclusion that in the case investigated the duration of the metabolic stage of the y-ac region of the sc8 chromosome is hereditarily determined by four factors:

  1. The biochemical nature of the y-ac region.
  2. Its position relative to the inert region.
  3. The condition in which it passed through meiosis in the parents.
  4. The presence in the nucleus of additional inert regions (e.g. in the Y-chromosome).

All these agencies, underlying the hereditary nature of the chromosome cycles, are probably of general significance. The chromosomal cycle is determined genetically, and also depends directly on environment (Darlington, 1937, 1942). In the cases which I investigated a fall of temperature to 14° C. and a rise to 30° C. acted appreciably on the heterochromatic condition of the 20ABC-1AB1 regions, with the loci y and ac, thus lengthening the metabolic stage of this region.

The regularities of heterochromatization, namely, (1) its increase in active regions adjoining the inert ones, (2) its suppression in inert regions inserted into active ones, (3) its varying length in active regions adjoining the inert ones, due to the latter's sensitivity, (4) its suppression at temperatures of 14 and 30° C., and (5) its increase in the progeny with the age of female parents, are all in full accord with the regularities of crossing-over (Plough, 1917, 1921; Bridges, 1927, 1929; Kikkawa, 1932; Mather, 1939).

We have seen why three different phenomena, heterochromatization, mosaicism and crossing-over, obey the same regularities: they are all underlain by a single universal process. This is the change of the chromosome cycle as a reaction to the change of genetical, developmental and environmental conditions.

Warburg (1938) and others proved the participation of different nucleotides in the constitution of the enzymes of cellular respiration. Ostern and his collaborators (1938) showed that the yeast nucleic acids may serve as precursors for the coenzyme muscle adenylic acid. These facts lead to the conclusion that the different specific nucleotides of the respiratory enzymes may have their source in nucleic acids.

On the other hand, other workers concluded that content and character of nucleic acids in the cytoplasm and nucleus depend on the cycle of the nucleus (Caspersson, 1941; Caspersson & Schultz, 1938, 1939; Brachet, 1937). According to Brachet there is a reciprocal relation between the ribose- and desoxyribose nucleic acids in the cell. As Schultz writes: 'the Janus molecule that is the gene, depending upon the material available, turns its synthesis on one face to the increase of the nucleic acid component, as at the prophases of mitosis; or conversely during the interphases, the protein component is synthesized' (Schultz, 1941).

These data, along with the results of our investigation, lead to the conclusion that the hereditary character of the metabolic process is determined by the hereditary character of the chromosome cycle.

CONCLUSIONS

1. Heterochromatization is a normal change in the chromosome cycle, which indicates a transition of chromosome regions to the 'mitotic' condition.

2. Individual chromosome regions pass through their cycle in a relatively independent way, displaying heterochromatization at a time when the main nuclear complex is in the resting condition.

3. Under definite conditions any chromosome region may become a heterochromatic one in a metabolic nucleus. By the time of metaphase the whole chromosome complex of the nucleus is reduced to this state because of the intracellular conditions.

4. The conditions of precocious heterochromatization are not the same for all chromosome regions. For sensitive inert regions and for active regions located in their close proximity, the slightest changes in the biochemical condition of the cell, of which we have intimate knowledge, are sufficient to change their cycle to either side. The cycle of active chromosome regions remote from the centromere in the metabolic nucleus is changed with much greater difficulty.

5. The nucleus responds in the same way to changes in environmental, developmental and genetical conditions, namely, by changing its cycle. In the cases investigated, the inert regions, including 20ABC, and the adjoining active regions, section 1AB1 with the loci y and ac, responded to the alteration of developmental conditions due to temperature, the introduction of an additional Y-chromosome, the direction of the cross, or the age of parents, by changing their cycle.

6. Changes in the cycle of the chromosome at the stage of metabolic nucleus are of particular importance for the organism as a whole. In mosaic lines, the reduction of the metabolic stage in a given chromosome region (1AB1, 3C2) taking place as a result of heterochromatization, effects the trend of the development of the character connected with a given region towards the recessive manifestation of the character.

7. Within a single tissue, the degree of heterochromatization of the same chromosome section (1AB1-20ABC, 3C2-101) varies according to the cell, displaying a mosaic picture. The percentage of heterochromatization in each separate mosaic line has a rather constant value.

8. The mosaic manifestation of a character is a result of the variability of the cycles of inert regions and of regions situated near them in chromosomes of the various cells of the same tissue.

9. The hereditary nature of the duration of the metabolic stage of a given chromosome section is determined by four agents: (1) its position in the chromosome with respect to the sensitive inert region, (2) its state during the meiosis in the parents, (3) the presence in the nucleus of additional inert regions (e.g. Y-chromosome), and (4) its biochemical nature.

10. The duration of the metabolic stage of the y-ac section in the sc8 chromosome in all the cases examined can, according to the percentage of heterochromatization, be expressed by the following series:

 sc8 >  sc8 >  sc8 >  sc8 > y ac v
—— —— —— —— ————
XY  Y y ac v  sc8 sc8

This series fully corresponds to the types of mosaicism (as determined for the respective categories of flies by Noujdin, 1944).

11. The nuclear cycle is a subtle hereditary intracellular mechanism, the duration of whose separate stages controls the course of the morphogenetic developmental processes. It is also the mechanism by means of which the cell nucleus responds to the varying environmental condition. The actual direction of development is determined by the real course of the hereditary nuclear and cellular cycles under definite developmental conditions.

12. Three phenomena, heterochromatization, crossing-over, and mosaicism, are thus found to obey the same regularities, being underlain by a common universal process—the variation of the chromosome cycle, as its response to varying developmental conditions

ACKNOWLEDGEMENT

The author is deeply indebted to Professor J. B. S. Haldane for looking through the manuscript and preparing it for publication.

REFERENCES