- Email: [email protected]
Ribosomal Deoxyribonucleic Acid Microchromosome
Chromosomal Localization of the Major Histocompatibility (B) Complex (MHC) and Its Expression in Chickens Aneuploid for the Major Histocompatibility Complex/Ribosomal Deoxyribonucleic Acid Microchromosome s. E. BLOOM Department of Poultry and Avian Sciences, Cornell University, Ithaca, New York 14853 W. E. BRILES and R. W. BRILES Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60115 M. E. DELANY and R. R. DIETERT
(Received for publication January 7, 1987) ABSTRACT By the cytogenetic method of trisomy mapping, the major histocompatibility complex (MHC) (B) was located on the microchromosome that also contains all of the ribosomal ribonucleic acid (rDNA) genes that are detected as a nucleolar organizer region. Crosses involving aneuploid chickens homozygous or heterozygous for particular B haplotypes yield an F, of disomic, trisomic, and tetrasomic offspring suitable for studies of gene dosage effects and gene regulation. Studies to date show that MHC genes are expressed on each chromosome in aneuploid cells unlike the rDNA gene cluster, which is regulated to produce only diploid levels of mature rRNA, Biological effects of extra MHC chromosome dosage range from altered cell surface content of glycoproteins to altered growth potential of chickens. In addition, enhanced MHC-encoded cell surface products may influence the progression of B-cell differentiation and cell population dynamics in the developing bursa of Fabricius. Recent research shows a possible mechanism to account for the formation of unequal products in meiosis within the rDNA and MHC gene clusters. Unequal recombinational events may be promoted in the meiotic process in trisomic chickens. (Key words: major histocompatibility complex, trisomy mapping, ribosomal ribonucleic acid genes, gene dosage, gene regulation) 1987 Poultry Science 66:782-789 INTRODUCTION
A basic consideration in the study of genes and gene actions is the chromosomal location of the genetic trait of interest and its relationship to other loci in the genome. For any unmapped gene in the chicken, there is the potential for it to reside on any one of 39 chromosomes (i.e., m = 39) most of which are very small elements often referred to as microchromosomes (Ohno, 1961; Owen, 1965). It is of particular interest to learn about the content and arrangement of genes on such small chromosomes and to see what role, if any, their products play in basic cell processes connected with growth and development. It is not a small challenge to the geneticist to obtain such information. Knowledge of linkage relationships can have value beyond the level of basic information. For example, a linked gene that has an easily detectable phenotype provides a means for screening individuals in a population for naturally occur-
ring variations for chromosome (and gene) copy number. Such a marker gene complex is present on the chromosome containing the major histocompatibility (B) complex in the chicken, that is, the ribosomal ribonucleic acid genes (rDNA) that are detected as a nucleolar organizer region (NOR). This has enabled us to detect, and use in breeding, MHC chromosome number variants called aneuploids (Bloom and Bacon, 1985). These animals that have extra chromosomes have also been very useful for investigating gene dosage effects in development and cellular mechanisms for the regulation of gene expression at the MHC and the rDNA. It is the purpose of this review to discuss the particular research with aneuploid chickens that has allowed us to 1) locate the MHC on a specific chromosome, 2) determine the linkage of the MHC to another gene complex, the rDNA, 3) modulate the MHC copy number for studies on gene regulation and biological effects of extra
782
Downloaded from http://ps.oxfordjournals.org/ at Boston University on June 27, 2015
Department of Poultry and Avian Sciences, Cornell University, Ithaca, New York 14853
SYMPOSIUM: AVIAN MAJOR HISTOCOMPATIBILITY COMPLEX
gene products, and 4) investigate the possible enhancement of unequal crossing over on the MHC/rDNA microchromosome in the aneuploid state. LINKAGE OF THE MAJOR HISTOCOMPATIBILITY COMPLEX AND RIBOSOMAL DEOXYRIBONUCLEIC ACID
TRISOMY MAPPING
MHC
Disomic
Trisomic
(I []
[] [] [] I] I] [] []
13
6
13
15
Tetrasomic
6 13 15 21
ASSAY: Blood typing FIG. 1. Concept of mapping genes using aneuploid individuals (trisomies and tetrasomics). The major histocompatibility (MHC) can be mapped by detection of animals with extra B haplotypes and identification of the extra chromosomes involved.
MHC resides on Chromosome 17 of the mouse (H-2) and Chromosome 6 of the human (HLA) (Van Someren etal., 1974; Klein, 1975). Three major subregions are known for the chicken MHC: B-L, B-F, and B-G. The former two regions have known mammalian counterparts (Hala et al., 1976; Pink et al., 1977). Previous research using classical approaches to establishing linkage failed to show any association of the MHC with known linkage groups in the chicken (Briles and Gilmour, 1979; Etches and Hawes, 1979). The rDNA encodes the 18S + 5.8S + 28S rRNA used in ribosome biogenesis (Nomura et al., 1984). The rRNA genes occur as tandem repeats at the chromosomal sites known as NOR. For each NOR there is a nucleolus formed, representing the collective transcriptional activity of the rDNA gene cluster and the associated process of ribosome biogenesis. It is the nucleolus that provides a simple and convenient phenotype to detect aneuploids for the chromosome containing the MHC and the rDNA alike. Acridine orange cytochemistry allows the unambiguous detection of nucleolar numbers within each nucleus of the cell (and therefore MHC/ rDNA chromosome numbers) in air-dried slide preparations (Muscarella et al., 1985). Feather pulp is a convenient source of cells from newly hatched and growing chickens (Sandnes, 1954; Macera and Bloom, 1981). Animal population sizes of 500 to 1,000 can be nucleoli typed in routine fashion within the context of a genetic selection program. The detection of individual chickens with extra nucleoli and additional B haplotypes provided the raw material for mapping by the use of an aneuploid series. In principle, one can map a gene by simply detecting additional gene products and then finding the particular extra chromosome involved. For the MHC, this means blood typing chickens to detect heterozygotes, and for the rDNA, detecting additional nucleoli per cell. While a single aneuploid chicken provided a starting point for mapping, the development of trisomic lines with multiple, segregating B haplotypes was needed to obtain the necessary data. Various test crosses were made involving five different haplotypes. In each generation, birds were both blood-typed and nucleoli-typed. It was possible, eventually, to establish two basic types of matings that yielded both different ratios and types of progeny. In the first, a bird with two nucleoli per cell and two haplotypes
Downloaded from http://ps.oxfordjournals.org/ at Boston University on June 27, 2015
The method that was ultimately successful for establishing the linkage of the MHC and rDNA is known as trisomy mapping (Figure 1). This method has been used widely in mapping genes in plants where aneuploids are common (Swanson, 1960; Brown, 1972). The aneuploid individuals that one needs for mapping include trisomies and tetrasomics; these terms indicate that cells have one and two extra chromosomes, respectively, for a particular element in the karyotype. In warm-blooded animals, viable aneuploids are rare principally because of the teratogenic effects of the extra gene dosage. For most of the systems studied, the aneuploidies involve fairly large chromosomes, so the genetic disturbance is great. Avian species are unique in that there are many very small chromosomes in the genome; therefore, aneuploids for these elements may have little or no deleterious biological consequences. Such is the case for trisomies of the MHC/rDNA chromosome in chickens (Macera and Bloom, 1981; Bloom and Bacon, 1985). The MHC of the chicken was discovered as a blood group system by Briles et al. (1950), and was shown to exert strong responses in skin grafts by Schierman and Nordskog (1961). This system is now considered homologous to the mammalian MHC (Pazderka et al, 1975). The
783
BLOOM ET AL.
784
Detecting linkage of the MHC and rDNA: Disomic x Trisomic cros3 nucleolus (©9 0 B-type
@§
6,13,15
15.21
Q Q© S © B
13,15
15,21 6,15
6,15, 21
13,15, 21
Trisomies 3 B-types 3 NOR
FIG. 2. Illustration of a linkage test using a dam with three B haplotypes and a diploid heterozygous sire. There is a 1:1 segregation in the F, of disomic to trisomic progeny and a correlation between the maximum nucleolus number per cell and the number of B haplotypes. MHC = Major histocompatibility complex; rDNA = ribosomal deoxyribonucleic acid; NOR = nucleolar organizer regions.
(e.g., B{5, B2i) was crossed with one having three nucleoli per cell and three haplotypes (e.g., B6, B 13 , fi'5) (Figure 2). In the second mating scheme, male and female birds having three nucleoli per cell and three haplotypes each were crossed (Table 1). Such matings gave highly reproducible results, consistent with a simple chromosome linkage model and Mendelian segregation of all chromosomes involved. A 1:1 ratio of disomic to trisomic types was obtained
CHROMOSOMAL LOCATION OF THE MAJOR HISTOCOMPATIBILITY COMPLEX AND RIBOSOMAL DEOXYRIBONUCLEIC ACID
Chromosomal localization of the MHC in chickens was established via its linkage to the NOR (Bloom etal., 1978; Bloom, 1979; Bloom and Bacon, 1985). The exact location of the NOR was demonstrated using silver nitrate cytochemistry (Goodpasture and Bloom, 1975). This technique resolves the precise location for the rDNA and also reveals transcriptionally active sites (Miller et al., 1976). Once the NOR was identified, we were able to establish the chromosomal status of birds with extra B haplotypes and thereby confirm that both the rDNA and MHC were located on a particular chromosome.
TABLE 1. Production of aneuploid chickens expressing two, three, and four different major histocompatibility complex haplotypes on erythrocytes Trisomic parents Sire
Dam
6/15/21 X 6/13/21
B Complex genotypes of offspring Disomic
No.
6/6
6/15 6/21 13/21 15/21 21/21
Totals: 'Can be 6/6/21 or 6/21/21.
5
10
Trisomic
No.
6/6/13 6/6/15 6/15/21 6/13/21 6/13/15 6/21/-1 13/21/21 13/15/21
1 1 1
1 3 5 2 2 16
Tetrasomic
No.
6/6/21/21 6/6/13/15 13/15/21/21 6/13/15/21
2 2 1 2
7
Downloaded from http://ps.oxfordjournals.org/ at Boston University on June 27, 2015
Disomies 2 B-types 2 NOR
6,13, , , 15
when one parent had three nucleoli per cell. A 1:2:1 ratio of disomic, trisomic, and tetrasomic types resulted from the crossing of two trisomic parental types. A key finding was the formation of the tetrasomic type with cells having four nucleoli per cell. In this category, individuals having four haplotypes (B6, B13, B15, B21) were also detected (Table 1). These crosses established the linkage relationship of the MHC to the rDNA and provided genetic information to suggest that the individuals with extra nucleoli and haplotypes were trisomies and tetrasomics. However, the necessary data were missing on the chromosomal status of these animals. Such information was eventually obtained with the use of a cytochemical technique having sufficient resolution to pinpoint the NOR on microchromosomes.
SYMPOSIUM: AVIAN MAJOR HISTOCOMPATIBILITY COMPLEX
MHC rRNA \s
w
FIG. 3. Proposed chromosome and linkage map for the microchromosome containing the major histocompatibility complex (MHC) and ribosomal ribonucleic acid genes (rRNA), detected as a nucleolar organizer region (NOR). The NOR occupies a majority of the long arm. The MHC occupies some or all of the region shown in brackets. (From Bloom and Bacon, 1985; Copyright 1985 by the American Genetic Association).
encoded within the short arm and proximal portion of the long arm and thus occupies a pericentromeric position. Further studies are needed to resolve this issue. MAJOR HISTOCOMPATIBILITY COMPLEX EXPRESSION IN ANEUPLOIDS
The aneuploid chicken strain provides an excellent model for study of the regulation of gene expression for both the MHC and rDNA gene clusters. This is interesting from both basic and applied standpoints. There is currently minimal information on mechanisms for the regulation of gene dosage in birds and the potentially special nature of gene regulation in microchromosomes. It is of interest and possibly of use to the breeder to know about gene locations and gene copy number variants of potential economic importance. Both the rDNA (involved in regulating protein synthesis) and the MHC (involved in immune regulation) are clearly of interest in this regard (Bloom, 1983). Crosses involving trisomic parents of known B haplotypes have provided the material for studies on genetic regulation; in the F( of this type of cross, the MHC/rDNA chromosome dosage series disomic, trisomic, and tetrasomic is produced (Table 1). At hatching, a 1:2:1 ratio of these three genotypes is observed; a majority of the aneuploid chickens can be reared to adulthood. Evidence from a number of lines of investigation indicates that both the MHC and rDNA sequences are transcribed on each microchromosome in aneuploid cells. For example, three and four nucleoli and silver-positive microchromosomes are seen in aneuploid cells. Also, there is expression of three and four different haplotypes on trisomic and tetrasomic erythrocytes, respectively. Therefore, chromosome inactivation of the type known for the mammalian sex chromosome system does not operate in the aneuploid chromosome state. However, evidence for regulation at the DNA level has been obtained for the ribosomal RNA genes. First, the amounts of mature 18S and 28S ribosomal RNA genes are not increased above the normal diploid level in aneuploid cells. Second, molecular analysis reveals that portions of the ribosomal RNA gene cluster are not transcribed in aneuploid cells. Evidence for regulation at the level of chromatin has been obtained using deoxyribonuclease I hypersensitivity and topoisomerase I assays, both of which detect ac-
Downloaded from http://ps.oxfordjournals.org/ at Boston University on June 27, 2015
A silver nitrate technique known as the AgAS method (also Ag-banding) (Bloom and Buss, 1969; Goodpasture and Bloom, 1975) was applied to chromosome preparations of animals having additional nucleoli and B haplotypes. The NOR was identified on the long arm of a medium-sized microchromosome, and just two such chromosomes were seen in normal disomic animals. In birds having three nucleoli and three B haplotypes, three silver-stained (Ag-NOR) microchromosomes were seen, confirming the suspected trisomic condition. Four Ag-NOR were visualized in chromosome preparations from birds having four nucleoli, thus confirming the tetrasomic genotype. Karyotype analysis revealed that the chromosome encoding the rDNA and MHC was a medium-sized microchromosome ranking about 16th in size. The chromosome is acrocentric with a discernible (but small) short arm (Bloom and Bacon, 1985). Southern blotting using a cloned chicken rDNA probe provided molecular confirmation of the aneuploid series (disomic, trisomic, and tetrasomic), and also gave information on the number of rDNA repeats in these genotypes (Muscarella et al., 1985). It is estimated that some 6,000 kilobases (kb) of DNA are arranged as approximately 145 rDNA repeats, and about 2,000 kb is an estimate of MHC size. These two gene clusters (and the centromere) probably occupy most, if not all, of the DNA in this microchromosome (Figure 3). Although the exact subchromosomal location for the MHC is unknown, it is likely that it resides on the long arm proximal to the centromere. Other candidate sites are the distal long arm region (beyond the rDNA), or perhaps the short arm. It is also possible that the MHC is
785
786
BLOOM ET AL. TABLE 2. Quantitative adsorption analysis of major histocompatibility complex-encoded on normal and aneuploid adult chicken erythrocytes
glycoproteins
Genotype of adsorbing cells
No. of animals
No. of adsorbing cells (X ± SD X 10 6 ) 1
Disomic Trisomic Tetrasomic
7 16 7
15.0 + 4.1;; 9.3 2.8 U 4.8 : 1.4C
B1$Bi5B15 B15B1SB1SB1S
Means with different superscripts are significantly different in a two sample t test (P<.001). 'Number of adsorbing cells that inhibit the hemagglutination reactivity of an anti-B 15 sera toward erythrocytes by 50%.
full range of MHC-encoded biological effects within the lymphoid and other systems of the chicken. BIOLOGICAL EFFECTS OF EXTRA MAJOR HISTOCOMPATIBILITY COMPLEX COPIES
It is of interest to ask what, if any, biological effects may result from the extra gene dosage in these animals with extra copies of the MHC/ rDNA chromosome. Also, since the rDNA is regulated in aneuploid cells, we can approach more closely an examination of MHC-related gene dosage effects. At the same time, it must be noted that 1) the MHC is itself a complex genetic region and 2) still other unknown DNA sequences could be a part of a gene dosage effect. A candidate for this sequence would be a region like the t complex in the mouse. No such region has yet been identified in the avian genome. However, our estimate is that most of the coding portion of the microchromosome is comprised of the rDNA and MHC sequences. We have searched for evidence of gene dosage effects both at the organismal and cellular levels. Little effect of MHC copy number on growth and development of the embryo in vivo has been observed. However, after hatching, a small but significant influence on the growth rate of trisomies has been seen (Bloom et al., 1985). Growth depression observed is maximally expressed between 10 to 14 weeks of age. Tetrasomic chicks have poor neonatal viability; many expire within the first two weeks after hatching. Survivors show a 30% reduction in body weight in the growing period compared to disomic control chickens. Aneuploid chickens do not display any obvious physical anomalies but tetrasomic chicks are noted to be shy and nervous upon handling. Necropsy reports reveal a high incidence of heart and kidney malforma-
Downloaded from http://ps.oxfordjournals.org/ at Boston University on June 27, 2015
tive versus inactive regions on the DNA (Muscarella et al., 1985; Muscarella et al., unpublished). Thus, it appears that sets of rRNA genes on each chromosome in the aneuploid cell are not activated for transcription. The situation for the MHC appears to be different. Using erythrocyte samples from homozygous B]5 disomic, trisomic, and tetrasomic chickens it has been possible to demonstrate increased adsorption power of the aneuploid cells toward a B -detecting alloantiserum (Delany etal., 1985). Indeed, there is a stepwise increase in adsorption capacity of cells across the three genotypes (Table 2). This indicates that there are additional glycoprotein sites per erythrocyte in the trisomic and tetrasomic animals that are encoded by MHC subregions. Evidence for enhanced MHC Class II product on aneuploid lymphocytes has recently been obtained. Using a mouse antichicken immune response antigen (la) monoclonal antibody (Ewert et al., 1984), it has been possible to show a stepwise increase in cell surface la on disomic, trisomic, and tetrasomic bursa-resident B lymphocytes, respectively (Delany et al., 1986). These results are indicative of normal levels of B-L expression resulting from each MHC-bearing chromosome in the aneuploid lymphocytes. The model that emerges from these studies is that of differential gene expression on the MHC/rDNA microchromosome. While subsets of ribosomal RNA genes are inactive on each chromosome in the aneuploid cell, MHC genes on each chromosome are transcribed at rates that result in enhanced amounts of cell surface product per cell in both trisomies and tetrasomics. These results suggest that the aneuploid chicken may in fact be a useful model for the study of MHC gene dosage effects. As a result, aneuploids may be used further in the search for the
B15Bls
SYMPOSIUM: AVIAN MAJOR HISTOCOMPATIBILITY COMPLEX
POSSIBILITIES FOR ENHANCING GENE COPY NUMBER SHIFTS IN CHICKENS
During the course of our screening of progeny from trisomic matings, deviations from expected nucleolar phenotypes have been observed. These are in the form of obvious polymorphisms for nucleolar sizes visualized by acridine orange cytochemistry. The most dramatic (and common) case is the presence of one large and one very small nucleolus in a diploid nucleus (with the normal phenotype there are two equal-sized nucleoli per cell). When such polymorphic diploid chickens are outcrossed to normal diploid birds, a segregation of the polymorphic nucleolus condition is seen in the F! generation, giving approximately a 1:1 ratio of normal to polymorphic animals.Furthermore, it has been possible to pinpoint the genesis of a polymorphic bird to a particular mating when at least one parent is a trisomic. From these observations we have developed the hypothesis that an increase or decrease in rDNA gene copy number underlies the nucleolar polymorphisms and that a shift in copy number can occur in a single meiosis. Variation for rDNA gene copy numbers is well known in other systems, particularly in Escherichia coli, Neurospora, Drosophila, and in cultured mammalian cells (Krider and Plaut, 1972; Miller et al, 1976; Tantravahi et al,
1981; Jinks-Robertson et al., 1983). Ribosomal DNA is modulated both somatically and in meiosis. Out-of-register pairing (ORP) between sister chromatids has been invoked as the initial step leading to the formation of chromosomes having increased or decreased gene copy numbers (Tartof, 1974; Peters, 1980). The rDNA region may be more susceptible to ORP since there are long stretches of repeated sequences, such that chromatid and chromosome pairing can be achieved at many sites. Findings from the trisomic system suggest enhanced susceptibility to the production of polymorphic (and unequal) products in meiosis. We hypothesize that ORP is enhanced in the formation of a trivalent in meiosis of trisomic animals and that this situation leads to altered rDNA gene copy numbers. These sequence changes are reflected as enlarged or decreased nucleolar sizes in chicken cells. The ORP system could effect crossing over within the MHC, particularly if (and it appears to be the case) the MHC and rDNA are tightly linked on the microchromosome. If the rDNA pairs out-of-register, the MHC might also be pulled slightly out of register. As there are blocks of related DNA sequences within the MHC (at least these have been identified in mammalian species)-as in the case of Class I DNA-pairing and crossing over would still occur over related or homologous sequences. However, the results of crossing over following ORP within the MHC would be the formation of chromosomes with additions or deletions of MHC sequences. In this regard, evidence for a duplication of a portion of the B-G subregion of the chicken MHC has recently been presented (Briles et al., 1982; Miller et al., 1986). These hypotheses concerning MHC recombinational mechanisms await testing in future studies.
SUMMARY AND CONCLUSIONS
Studies to date show clearly the linkage of the MHC to the rDNA gene cluster in the chicken genome. These two regions map to a mediumsized microchromosome (about Pair Number 16); this particular chromosome can be identified in metaphase preparations using the Ag-AS banding methodology. This same technique resolves the rRNA gene cluster as an NOR on the long arm of the microchromosome. The precise location of the MHC on this element has yet to be demonstrated. The most likely site for the MHC is on the distal long arm region next to the NOR.
Downloaded from http://ps.oxfordjournals.org/ at Boston University on June 27, 2015
tions in the aneuploids. Reproductive parameters appear normal in adult male and female trisomies. Tetrasomic females, however, lay few eggs. Interestingly, although tetrasomic males do not respond well in artificial insemination, they are quite fertile in the pen mating situation. Recently we have detected gene dosage effects within the developing immune system of aneuploid chicks. These include alterations in bursal cell populations and in monocyte-macrophage properties. Flow cytometric analysis of la antibody-tagged B-cells revealed a shift in the proportion of large vs. small cell populations toward increasing numbers of small cells in the aneuploid neonatal bursa (Delany et al., 1986). Alterations in monocyte-macrophage development and functions include a lower yield of adherent macrophages in tetrasomic birds, depressed incidence of phagocytic activity by aneuploid macrophages toward uncoated erythrocytes, and enhanced chemotactic responses by tetrasomic monocytes to f-met-leu-phe and Enterobacter cloacae supernatant (Qureshi et al., 1986).
787
BLOOM ET AL.
788
ACKNOWLEDGMENTS
The authors are grateful to Douglas Gilmour, Larry Bacon, Donna Muscarella, and Louis Schierman for encouragement and helpful discussions. The editorial assistance of Diane Coif and Julie Ruocco is greatly appreciated. REFERENCES Bloom, S. E., 1979. Linkage relationships: Chicken. In: Inbred and Genetically Defined Strains of Laboratory Animals, Part 2. P. L. Altman and D. D. Katz, ed. Fed. Am. Soc. Exp. Biol., Bethesda, MD. Bloom, S. E., 1983. New approaches for the further genetic improvement of poultry. Pages 5-8 in: Proc. Cornell Nutr. Conf., Ithaca, NY.
Bloom, S. E., and L. D. Bacon, 1985. Linkage of the major histocompatibility (B) complex and the nucleolar organizer in the chicken: Assignment to a microchromosome. J. Hered. 76:146-154. Bloom, S. E., and E. G. Buss, 1969. Ammoniacal silver staining of embryonic chicken cells and chromosomes. Poultry Sci. 48:1114-1116. Bloom, S. E., P. Shalit, and L. D. Bacon, 1978. Chromosomal localization of nucleolus organizers in the chicken. Genetics 88:S13. (Abstr.) Bloom, S. E., E. M. Willand, and M. E. Delany, 1985. Development potential of chickens aneuploid for the rDNA/MHC microchromosome. Genetics 110:S3. (Abstr.) Briles, W. E., R. W. Briles, and R. E. Taffs, 1982. An apparent recombinant within the B-G region of the B complex. Poultry Sci. 61:1425-1426 Briles, W. E., and D. G. Gilmour, 1979. Blood group systems: Chicken. Part 1. Erythrocyte alloantigen characteristics. Page 650 in: Inbred and Genetically Defined Strains of Laboratory Animals. Part 2. P. L. Altman, and D. D. Katz, ed. Fed. Am. Soc. Exp. Biol. Handbook III. Briles, W. E., W. H. McGibbon, and M. R. Irwin, 1950. On multiple alleles effecting cellular antigens in the chicken. Genetics 35:633-652. Brown, W. V., 1972. Textbook of Cytogenetics. C. V. Mosby Co., St. Louis, MO. Delany, M. E., W. E. Briles, R. W. Briles, R. R. Dietert, E. M. Willand, and S. E. Bloom, 1985. Cell surface expression of major histocompatibility complex-encoded glycoprotein antigens in normal and aneuploid chickens. Genetics 110:S27. (Abstr.) Delany, M. E., R. R. Dietert, and S. E. Bloom, 1986. Increased membrane expression of la antigen and altered cell populations in bursa of aneuploid chickens. Poultry Sci. 65(Suppl 1):33. (Abstr.) Etches, R. J., and R. O. Hawes, 1979. Linkage relationship: Chicken. Part IV. Genes demonstrating independent segregation. Pages 628-637 in: Inbred and Genetically Defined Strains of Laboratory Animals. P. L. Altman and D. D. Katz, ed. Fed. Am. Soc. Exp. Biol., Bethesda, MD. Ewert, D. L., M. S. Munchus, Chen-Lo H. Chen, and M. D. Cooper, 1984. Analysis of structural properties and cellular distribution of avian la antigen by using monoclonal antibody to monomorphic determinants. J. Immunol. 132:2524-2530. Goodpasture, C , and S. E. Bloom, 1975. Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining. Chromosoma 53:37-50. Hala, K., M. Vilhelmova, and J. Hartmanova, 1976. Probable crossing-over in the B blood group of chickens. Immunogenetics 3:97-104. Jinks-Robertson, S., R. L. Gourse, and M. Nomura, 1983. Expression of rRNA and tRNA genes in Escherichia coli: evidence for feedback regulation by products of rRNA operons. Cell 33:865-876. Klein, J., 1975. Biology of the Mouse Histocompatibility Complex. Springer Verlag, Berlin, BRD. Krider, H. M., and W. Plaut, 1972. Studies on nucleolar RNA synthesis in Drosophila melanogaster I: the relationship between number of nucleolar organizers and rate of synthesis. J. Cell Sci. 11:675-687. Macera, M. J., and S. E. Bloom, 1981. Ultrastructural studies of the nucleoli in diploid and trisomic chickens. J. Hered. 72:249-252.
Downloaded from http://ps.oxfordjournals.org/ at Boston University on June 27, 2015
The discovery of aneuploids for the MHC not only facilitated its mapping but also aided studies of gene regulation and gene dosage effects in development. The picture emerging is that of differential gene expression of MHC and rDNA on the chromosome. Although the rDNA is regulated on each chromosome, the MHC appears to be expressed and we find extra cell surface products on both erythrocytes and B lymphocytes. Thus, we do not see evidence for whole chromosome inactivation to achieve dosage regulation in aneuploid avian cells. These and other considerations support the use of aneuploid chickens as a model to examine MHC gene dosage effects on developing systems in the chicken. Studies with this and other systems, such as congenic lines, will allow the dissection of the range of biologic influence of the MHC as a gene cluster. Such studies have already implicated the MHC as one factor influencing hatchability, postnatal growth potential, and even reproductive performance. The MHC influence, therefore, apparently extends beyond its actions in regulating the immune response to other traits of economic importance. It may not be entirely unreasonable to speculate at this stage that the MHC is a major fitness complex for Gallus domesticus and other species. This effect on fitness, related to the activity of the MHC, is hypothesized to consist of alterations in cell differentiation and tissue formations. Finally, the chicken MHC is on the map. It now stands along side of the mouse H-2 complex on Chromosome 17 and the human HLA on Chromosome 6. It will be of great interest to determine the extent of sequence and chromosome homology among species and to learn of the most highly conserved vs. highly evolving MHC sequences and functions.
SYMPOSIUM: AVIAN MAJOR HISTOCOMPATIBILITY COMPLEX
A. Ziegler, 1977. A three-locus model for the chicken major histocompatibility complex. Immunogenetics 5:203-216. Qureshi, M. A., S. E. Bloom, and R. R. Dietert, 1986. Macrophage function in chickens having increased copy number. Poultry Science 65(Suppl. I): 108. (Abstr.) Sandnes, G. C , 1954. A new technique for the study of avian chromosomes. Science 119:508-509. Schierman, L. W., and A. W. Nordskog, 1961. Relationship of blood type to histocompatibility in chickens. Science 134:1008-1009 Swanson, C. P., 1960. Cytology and Cytogenetics, 3rd ed. Prentice Hall, Inc., Englewood Cliffs, NJ. Tantravahi, U., R. V. Guntaka, B. F. Erlanger, and O. J. Miller, 1981. Amplified ribosomal RNA genes in a rat hepatoma cell line are enriched in 5-methyIcytosine. Proc. Natl. Acad. Sci. 78:489^193. Tartof, K. D., 1974. Unequal meitotic sister chromatid exchange as the mechanism of ribosomal RNA gene magnification. Proc. Natl. Acad. Sci. 71:1272-1276. Van Someren, H., A. Westerveld, A. Hagemaijer, J. R. Mees, K. Meera, and O. B. Zaalberg, 1974. Human antigen and enzyme markers in man-Chinese hamster somatic cell hybrids: evidence for synteny between the HL-A, PGMe, ME1, and IPO-B loci. Proc. Natl. Acad. Sci. 71:962-965.
Downloaded from http://ps.oxfordjournals.org/ at Boston University on June 27, 2015
Miller, M. M., Goto, R., and W. E. Briles, 1986. Biochemical evidence for recombination within the B-G region of the chicken major histocompatibility complex. Poultry Sci. 65(Suppl. 1):94. (Abstr.) Miller, O. J., D. A. Miller, V. G. Dev, R. Tantravahi, and C M . Croce, 1976. Expression of human and suppression of mouse nucleolus organizer activity in mousehuman somatic cell hybrids. Proc. Natl. Acad. Sci. 73:4531-4535. Muscarella, D. E., V. M. Vogt, and S. E. Bloom, 1985. The ribosomal RNA gene cluster in aneuploid chickens: evidence for increased gene dosage and regulation of gene expression. J. Cell Biol. 101:1749-1756. Nomura, M. R., L. Gourse, andG. Baughman, 1984. Regulation of the synthesis of ribosomes and ribosomal components. Annu. Rev. Biochem. 53:75-117. Ohno, S., 1961. Sex chromosomes and microchromosomes of Gallus domesticus. Chromosoma 11:484-498. Owen, J.J.T., 1965. Karyotype studies on Gallus domesticus. Chromosoma 16:601-608. Pazderka, F., B. M. Longenecker, G.R.J. Law, and R. F. Ruth, 1975. The major histocompatibility complex of the chicken. Immunogenetics 2:101-130. Petes, T. D., 1980. Unequal miotic recombination within tandem arrays of yeast ribosomal DNA genes. Cell 19:765-774. Pink, J.R.L., W. Droege, K. Hala, V. C. Miggiano, and
789
(Received for publication January 7, 1987) ABSTRACT By the cytogenetic method of trisomy mapping, the major histocompatibility complex (MHC) (B) was located on the microchromosome that also contains all of the ribosomal ribonucleic acid (rDNA) genes that are detected as a nucleolar organizer region. Crosses involving aneuploid chickens homozygous or heterozygous for particular B haplotypes yield an F, of disomic, trisomic, and tetrasomic offspring suitable for studies of gene dosage effects and gene regulation. Studies to date show that MHC genes are expressed on each chromosome in aneuploid cells unlike the rDNA gene cluster, which is regulated to produce only diploid levels of mature rRNA, Biological effects of extra MHC chromosome dosage range from altered cell surface content of glycoproteins to altered growth potential of chickens. In addition, enhanced MHC-encoded cell surface products may influence the progression of B-cell differentiation and cell population dynamics in the developing bursa of Fabricius. Recent research shows a possible mechanism to account for the formation of unequal products in meiosis within the rDNA and MHC gene clusters. Unequal recombinational events may be promoted in the meiotic process in trisomic chickens. (Key words: major histocompatibility complex, trisomy mapping, ribosomal ribonucleic acid genes, gene dosage, gene regulation) 1987 Poultry Science 66:782-789 INTRODUCTION
A basic consideration in the study of genes and gene actions is the chromosomal location of the genetic trait of interest and its relationship to other loci in the genome. For any unmapped gene in the chicken, there is the potential for it to reside on any one of 39 chromosomes (i.e., m = 39) most of which are very small elements often referred to as microchromosomes (Ohno, 1961; Owen, 1965). It is of particular interest to learn about the content and arrangement of genes on such small chromosomes and to see what role, if any, their products play in basic cell processes connected with growth and development. It is not a small challenge to the geneticist to obtain such information. Knowledge of linkage relationships can have value beyond the level of basic information. For example, a linked gene that has an easily detectable phenotype provides a means for screening individuals in a population for naturally occur-
ring variations for chromosome (and gene) copy number. Such a marker gene complex is present on the chromosome containing the major histocompatibility (B) complex in the chicken, that is, the ribosomal ribonucleic acid genes (rDNA) that are detected as a nucleolar organizer region (NOR). This has enabled us to detect, and use in breeding, MHC chromosome number variants called aneuploids (Bloom and Bacon, 1985). These animals that have extra chromosomes have also been very useful for investigating gene dosage effects in development and cellular mechanisms for the regulation of gene expression at the MHC and the rDNA. It is the purpose of this review to discuss the particular research with aneuploid chickens that has allowed us to 1) locate the MHC on a specific chromosome, 2) determine the linkage of the MHC to another gene complex, the rDNA, 3) modulate the MHC copy number for studies on gene regulation and biological effects of extra
782
Downloaded from http://ps.oxfordjournals.org/ at Boston University on June 27, 2015
Department of Poultry and Avian Sciences, Cornell University, Ithaca, New York 14853
SYMPOSIUM: AVIAN MAJOR HISTOCOMPATIBILITY COMPLEX
gene products, and 4) investigate the possible enhancement of unequal crossing over on the MHC/rDNA microchromosome in the aneuploid state. LINKAGE OF THE MAJOR HISTOCOMPATIBILITY COMPLEX AND RIBOSOMAL DEOXYRIBONUCLEIC ACID
TRISOMY MAPPING
MHC
Disomic
Trisomic
(I []
[] [] [] I] I] [] []
13
6
13
15
Tetrasomic
6 13 15 21
ASSAY: Blood typing FIG. 1. Concept of mapping genes using aneuploid individuals (trisomies and tetrasomics). The major histocompatibility (MHC) can be mapped by detection of animals with extra B haplotypes and identification of the extra chromosomes involved.
MHC resides on Chromosome 17 of the mouse (H-2) and Chromosome 6 of the human (HLA) (Van Someren etal., 1974; Klein, 1975). Three major subregions are known for the chicken MHC: B-L, B-F, and B-G. The former two regions have known mammalian counterparts (Hala et al., 1976; Pink et al., 1977). Previous research using classical approaches to establishing linkage failed to show any association of the MHC with known linkage groups in the chicken (Briles and Gilmour, 1979; Etches and Hawes, 1979). The rDNA encodes the 18S + 5.8S + 28S rRNA used in ribosome biogenesis (Nomura et al., 1984). The rRNA genes occur as tandem repeats at the chromosomal sites known as NOR. For each NOR there is a nucleolus formed, representing the collective transcriptional activity of the rDNA gene cluster and the associated process of ribosome biogenesis. It is the nucleolus that provides a simple and convenient phenotype to detect aneuploids for the chromosome containing the MHC and the rDNA alike. Acridine orange cytochemistry allows the unambiguous detection of nucleolar numbers within each nucleus of the cell (and therefore MHC/ rDNA chromosome numbers) in air-dried slide preparations (Muscarella et al., 1985). Feather pulp is a convenient source of cells from newly hatched and growing chickens (Sandnes, 1954; Macera and Bloom, 1981). Animal population sizes of 500 to 1,000 can be nucleoli typed in routine fashion within the context of a genetic selection program. The detection of individual chickens with extra nucleoli and additional B haplotypes provided the raw material for mapping by the use of an aneuploid series. In principle, one can map a gene by simply detecting additional gene products and then finding the particular extra chromosome involved. For the MHC, this means blood typing chickens to detect heterozygotes, and for the rDNA, detecting additional nucleoli per cell. While a single aneuploid chicken provided a starting point for mapping, the development of trisomic lines with multiple, segregating B haplotypes was needed to obtain the necessary data. Various test crosses were made involving five different haplotypes. In each generation, birds were both blood-typed and nucleoli-typed. It was possible, eventually, to establish two basic types of matings that yielded both different ratios and types of progeny. In the first, a bird with two nucleoli per cell and two haplotypes
Downloaded from http://ps.oxfordjournals.org/ at Boston University on June 27, 2015
The method that was ultimately successful for establishing the linkage of the MHC and rDNA is known as trisomy mapping (Figure 1). This method has been used widely in mapping genes in plants where aneuploids are common (Swanson, 1960; Brown, 1972). The aneuploid individuals that one needs for mapping include trisomies and tetrasomics; these terms indicate that cells have one and two extra chromosomes, respectively, for a particular element in the karyotype. In warm-blooded animals, viable aneuploids are rare principally because of the teratogenic effects of the extra gene dosage. For most of the systems studied, the aneuploidies involve fairly large chromosomes, so the genetic disturbance is great. Avian species are unique in that there are many very small chromosomes in the genome; therefore, aneuploids for these elements may have little or no deleterious biological consequences. Such is the case for trisomies of the MHC/rDNA chromosome in chickens (Macera and Bloom, 1981; Bloom and Bacon, 1985). The MHC of the chicken was discovered as a blood group system by Briles et al. (1950), and was shown to exert strong responses in skin grafts by Schierman and Nordskog (1961). This system is now considered homologous to the mammalian MHC (Pazderka et al, 1975). The
783
BLOOM ET AL.
784
Detecting linkage of the MHC and rDNA: Disomic x Trisomic cros3 nucleolus (©9 0 B-type
@§
6,13,15
15.21
Q Q© S © B
13,15
15,21 6,15
6,15, 21
13,15, 21
Trisomies 3 B-types 3 NOR
FIG. 2. Illustration of a linkage test using a dam with three B haplotypes and a diploid heterozygous sire. There is a 1:1 segregation in the F, of disomic to trisomic progeny and a correlation between the maximum nucleolus number per cell and the number of B haplotypes. MHC = Major histocompatibility complex; rDNA = ribosomal deoxyribonucleic acid; NOR = nucleolar organizer regions.
(e.g., B{5, B2i) was crossed with one having three nucleoli per cell and three haplotypes (e.g., B6, B 13 , fi'5) (Figure 2). In the second mating scheme, male and female birds having three nucleoli per cell and three haplotypes each were crossed (Table 1). Such matings gave highly reproducible results, consistent with a simple chromosome linkage model and Mendelian segregation of all chromosomes involved. A 1:1 ratio of disomic to trisomic types was obtained
CHROMOSOMAL LOCATION OF THE MAJOR HISTOCOMPATIBILITY COMPLEX AND RIBOSOMAL DEOXYRIBONUCLEIC ACID
Chromosomal localization of the MHC in chickens was established via its linkage to the NOR (Bloom etal., 1978; Bloom, 1979; Bloom and Bacon, 1985). The exact location of the NOR was demonstrated using silver nitrate cytochemistry (Goodpasture and Bloom, 1975). This technique resolves the precise location for the rDNA and also reveals transcriptionally active sites (Miller et al., 1976). Once the NOR was identified, we were able to establish the chromosomal status of birds with extra B haplotypes and thereby confirm that both the rDNA and MHC were located on a particular chromosome.
TABLE 1. Production of aneuploid chickens expressing two, three, and four different major histocompatibility complex haplotypes on erythrocytes Trisomic parents Sire
Dam
6/15/21 X 6/13/21
B Complex genotypes of offspring Disomic
No.
6/6
6/15 6/21 13/21 15/21 21/21
Totals: 'Can be 6/6/21 or 6/21/21.
5
10
Trisomic
No.
6/6/13 6/6/15 6/15/21 6/13/21 6/13/15 6/21/-1 13/21/21 13/15/21
1 1 1
1 3 5 2 2 16
Tetrasomic
No.
6/6/21/21 6/6/13/15 13/15/21/21 6/13/15/21
2 2 1 2
7
Downloaded from http://ps.oxfordjournals.org/ at Boston University on June 27, 2015
Disomies 2 B-types 2 NOR
6,13, , , 15
when one parent had three nucleoli per cell. A 1:2:1 ratio of disomic, trisomic, and tetrasomic types resulted from the crossing of two trisomic parental types. A key finding was the formation of the tetrasomic type with cells having four nucleoli per cell. In this category, individuals having four haplotypes (B6, B13, B15, B21) were also detected (Table 1). These crosses established the linkage relationship of the MHC to the rDNA and provided genetic information to suggest that the individuals with extra nucleoli and haplotypes were trisomies and tetrasomics. However, the necessary data were missing on the chromosomal status of these animals. Such information was eventually obtained with the use of a cytochemical technique having sufficient resolution to pinpoint the NOR on microchromosomes.
SYMPOSIUM: AVIAN MAJOR HISTOCOMPATIBILITY COMPLEX
MHC rRNA \s
w
FIG. 3. Proposed chromosome and linkage map for the microchromosome containing the major histocompatibility complex (MHC) and ribosomal ribonucleic acid genes (rRNA), detected as a nucleolar organizer region (NOR). The NOR occupies a majority of the long arm. The MHC occupies some or all of the region shown in brackets. (From Bloom and Bacon, 1985; Copyright 1985 by the American Genetic Association).
encoded within the short arm and proximal portion of the long arm and thus occupies a pericentromeric position. Further studies are needed to resolve this issue. MAJOR HISTOCOMPATIBILITY COMPLEX EXPRESSION IN ANEUPLOIDS
The aneuploid chicken strain provides an excellent model for study of the regulation of gene expression for both the MHC and rDNA gene clusters. This is interesting from both basic and applied standpoints. There is currently minimal information on mechanisms for the regulation of gene dosage in birds and the potentially special nature of gene regulation in microchromosomes. It is of interest and possibly of use to the breeder to know about gene locations and gene copy number variants of potential economic importance. Both the rDNA (involved in regulating protein synthesis) and the MHC (involved in immune regulation) are clearly of interest in this regard (Bloom, 1983). Crosses involving trisomic parents of known B haplotypes have provided the material for studies on genetic regulation; in the F( of this type of cross, the MHC/rDNA chromosome dosage series disomic, trisomic, and tetrasomic is produced (Table 1). At hatching, a 1:2:1 ratio of these three genotypes is observed; a majority of the aneuploid chickens can be reared to adulthood. Evidence from a number of lines of investigation indicates that both the MHC and rDNA sequences are transcribed on each microchromosome in aneuploid cells. For example, three and four nucleoli and silver-positive microchromosomes are seen in aneuploid cells. Also, there is expression of three and four different haplotypes on trisomic and tetrasomic erythrocytes, respectively. Therefore, chromosome inactivation of the type known for the mammalian sex chromosome system does not operate in the aneuploid chromosome state. However, evidence for regulation at the DNA level has been obtained for the ribosomal RNA genes. First, the amounts of mature 18S and 28S ribosomal RNA genes are not increased above the normal diploid level in aneuploid cells. Second, molecular analysis reveals that portions of the ribosomal RNA gene cluster are not transcribed in aneuploid cells. Evidence for regulation at the level of chromatin has been obtained using deoxyribonuclease I hypersensitivity and topoisomerase I assays, both of which detect ac-
Downloaded from http://ps.oxfordjournals.org/ at Boston University on June 27, 2015
A silver nitrate technique known as the AgAS method (also Ag-banding) (Bloom and Buss, 1969; Goodpasture and Bloom, 1975) was applied to chromosome preparations of animals having additional nucleoli and B haplotypes. The NOR was identified on the long arm of a medium-sized microchromosome, and just two such chromosomes were seen in normal disomic animals. In birds having three nucleoli and three B haplotypes, three silver-stained (Ag-NOR) microchromosomes were seen, confirming the suspected trisomic condition. Four Ag-NOR were visualized in chromosome preparations from birds having four nucleoli, thus confirming the tetrasomic genotype. Karyotype analysis revealed that the chromosome encoding the rDNA and MHC was a medium-sized microchromosome ranking about 16th in size. The chromosome is acrocentric with a discernible (but small) short arm (Bloom and Bacon, 1985). Southern blotting using a cloned chicken rDNA probe provided molecular confirmation of the aneuploid series (disomic, trisomic, and tetrasomic), and also gave information on the number of rDNA repeats in these genotypes (Muscarella et al., 1985). It is estimated that some 6,000 kilobases (kb) of DNA are arranged as approximately 145 rDNA repeats, and about 2,000 kb is an estimate of MHC size. These two gene clusters (and the centromere) probably occupy most, if not all, of the DNA in this microchromosome (Figure 3). Although the exact subchromosomal location for the MHC is unknown, it is likely that it resides on the long arm proximal to the centromere. Other candidate sites are the distal long arm region (beyond the rDNA), or perhaps the short arm. It is also possible that the MHC is
785
786
BLOOM ET AL. TABLE 2. Quantitative adsorption analysis of major histocompatibility complex-encoded on normal and aneuploid adult chicken erythrocytes
glycoproteins
Genotype of adsorbing cells
No. of animals
No. of adsorbing cells (X ± SD X 10 6 ) 1
Disomic Trisomic Tetrasomic
7 16 7
15.0 + 4.1;; 9.3 2.8 U 4.8 : 1.4C
B1$Bi5B15 B15B1SB1SB1S
Means with different superscripts are significantly different in a two sample t test (P<.001). 'Number of adsorbing cells that inhibit the hemagglutination reactivity of an anti-B 15 sera toward erythrocytes by 50%.
full range of MHC-encoded biological effects within the lymphoid and other systems of the chicken. BIOLOGICAL EFFECTS OF EXTRA MAJOR HISTOCOMPATIBILITY COMPLEX COPIES
It is of interest to ask what, if any, biological effects may result from the extra gene dosage in these animals with extra copies of the MHC/ rDNA chromosome. Also, since the rDNA is regulated in aneuploid cells, we can approach more closely an examination of MHC-related gene dosage effects. At the same time, it must be noted that 1) the MHC is itself a complex genetic region and 2) still other unknown DNA sequences could be a part of a gene dosage effect. A candidate for this sequence would be a region like the t complex in the mouse. No such region has yet been identified in the avian genome. However, our estimate is that most of the coding portion of the microchromosome is comprised of the rDNA and MHC sequences. We have searched for evidence of gene dosage effects both at the organismal and cellular levels. Little effect of MHC copy number on growth and development of the embryo in vivo has been observed. However, after hatching, a small but significant influence on the growth rate of trisomies has been seen (Bloom et al., 1985). Growth depression observed is maximally expressed between 10 to 14 weeks of age. Tetrasomic chicks have poor neonatal viability; many expire within the first two weeks after hatching. Survivors show a 30% reduction in body weight in the growing period compared to disomic control chickens. Aneuploid chickens do not display any obvious physical anomalies but tetrasomic chicks are noted to be shy and nervous upon handling. Necropsy reports reveal a high incidence of heart and kidney malforma-
Downloaded from http://ps.oxfordjournals.org/ at Boston University on June 27, 2015
tive versus inactive regions on the DNA (Muscarella et al., 1985; Muscarella et al., unpublished). Thus, it appears that sets of rRNA genes on each chromosome in the aneuploid cell are not activated for transcription. The situation for the MHC appears to be different. Using erythrocyte samples from homozygous B]5 disomic, trisomic, and tetrasomic chickens it has been possible to demonstrate increased adsorption power of the aneuploid cells toward a B -detecting alloantiserum (Delany etal., 1985). Indeed, there is a stepwise increase in adsorption capacity of cells across the three genotypes (Table 2). This indicates that there are additional glycoprotein sites per erythrocyte in the trisomic and tetrasomic animals that are encoded by MHC subregions. Evidence for enhanced MHC Class II product on aneuploid lymphocytes has recently been obtained. Using a mouse antichicken immune response antigen (la) monoclonal antibody (Ewert et al., 1984), it has been possible to show a stepwise increase in cell surface la on disomic, trisomic, and tetrasomic bursa-resident B lymphocytes, respectively (Delany et al., 1986). These results are indicative of normal levels of B-L expression resulting from each MHC-bearing chromosome in the aneuploid lymphocytes. The model that emerges from these studies is that of differential gene expression on the MHC/rDNA microchromosome. While subsets of ribosomal RNA genes are inactive on each chromosome in the aneuploid cell, MHC genes on each chromosome are transcribed at rates that result in enhanced amounts of cell surface product per cell in both trisomies and tetrasomics. These results suggest that the aneuploid chicken may in fact be a useful model for the study of MHC gene dosage effects. As a result, aneuploids may be used further in the search for the
B15Bls
SYMPOSIUM: AVIAN MAJOR HISTOCOMPATIBILITY COMPLEX
POSSIBILITIES FOR ENHANCING GENE COPY NUMBER SHIFTS IN CHICKENS
During the course of our screening of progeny from trisomic matings, deviations from expected nucleolar phenotypes have been observed. These are in the form of obvious polymorphisms for nucleolar sizes visualized by acridine orange cytochemistry. The most dramatic (and common) case is the presence of one large and one very small nucleolus in a diploid nucleus (with the normal phenotype there are two equal-sized nucleoli per cell). When such polymorphic diploid chickens are outcrossed to normal diploid birds, a segregation of the polymorphic nucleolus condition is seen in the F! generation, giving approximately a 1:1 ratio of normal to polymorphic animals.Furthermore, it has been possible to pinpoint the genesis of a polymorphic bird to a particular mating when at least one parent is a trisomic. From these observations we have developed the hypothesis that an increase or decrease in rDNA gene copy number underlies the nucleolar polymorphisms and that a shift in copy number can occur in a single meiosis. Variation for rDNA gene copy numbers is well known in other systems, particularly in Escherichia coli, Neurospora, Drosophila, and in cultured mammalian cells (Krider and Plaut, 1972; Miller et al, 1976; Tantravahi et al,
1981; Jinks-Robertson et al., 1983). Ribosomal DNA is modulated both somatically and in meiosis. Out-of-register pairing (ORP) between sister chromatids has been invoked as the initial step leading to the formation of chromosomes having increased or decreased gene copy numbers (Tartof, 1974; Peters, 1980). The rDNA region may be more susceptible to ORP since there are long stretches of repeated sequences, such that chromatid and chromosome pairing can be achieved at many sites. Findings from the trisomic system suggest enhanced susceptibility to the production of polymorphic (and unequal) products in meiosis. We hypothesize that ORP is enhanced in the formation of a trivalent in meiosis of trisomic animals and that this situation leads to altered rDNA gene copy numbers. These sequence changes are reflected as enlarged or decreased nucleolar sizes in chicken cells. The ORP system could effect crossing over within the MHC, particularly if (and it appears to be the case) the MHC and rDNA are tightly linked on the microchromosome. If the rDNA pairs out-of-register, the MHC might also be pulled slightly out of register. As there are blocks of related DNA sequences within the MHC (at least these have been identified in mammalian species)-as in the case of Class I DNA-pairing and crossing over would still occur over related or homologous sequences. However, the results of crossing over following ORP within the MHC would be the formation of chromosomes with additions or deletions of MHC sequences. In this regard, evidence for a duplication of a portion of the B-G subregion of the chicken MHC has recently been presented (Briles et al., 1982; Miller et al., 1986). These hypotheses concerning MHC recombinational mechanisms await testing in future studies.
SUMMARY AND CONCLUSIONS
Studies to date show clearly the linkage of the MHC to the rDNA gene cluster in the chicken genome. These two regions map to a mediumsized microchromosome (about Pair Number 16); this particular chromosome can be identified in metaphase preparations using the Ag-AS banding methodology. This same technique resolves the rRNA gene cluster as an NOR on the long arm of the microchromosome. The precise location of the MHC on this element has yet to be demonstrated. The most likely site for the MHC is on the distal long arm region next to the NOR.
Downloaded from http://ps.oxfordjournals.org/ at Boston University on June 27, 2015
tions in the aneuploids. Reproductive parameters appear normal in adult male and female trisomies. Tetrasomic females, however, lay few eggs. Interestingly, although tetrasomic males do not respond well in artificial insemination, they are quite fertile in the pen mating situation. Recently we have detected gene dosage effects within the developing immune system of aneuploid chicks. These include alterations in bursal cell populations and in monocyte-macrophage properties. Flow cytometric analysis of la antibody-tagged B-cells revealed a shift in the proportion of large vs. small cell populations toward increasing numbers of small cells in the aneuploid neonatal bursa (Delany et al., 1986). Alterations in monocyte-macrophage development and functions include a lower yield of adherent macrophages in tetrasomic birds, depressed incidence of phagocytic activity by aneuploid macrophages toward uncoated erythrocytes, and enhanced chemotactic responses by tetrasomic monocytes to f-met-leu-phe and Enterobacter cloacae supernatant (Qureshi et al., 1986).
787
BLOOM ET AL.
788
ACKNOWLEDGMENTS
The authors are grateful to Douglas Gilmour, Larry Bacon, Donna Muscarella, and Louis Schierman for encouragement and helpful discussions. The editorial assistance of Diane Coif and Julie Ruocco is greatly appreciated. REFERENCES Bloom, S. E., 1979. Linkage relationships: Chicken. In: Inbred and Genetically Defined Strains of Laboratory Animals, Part 2. P. L. Altman and D. D. Katz, ed. Fed. Am. Soc. Exp. Biol., Bethesda, MD. Bloom, S. E., 1983. New approaches for the further genetic improvement of poultry. Pages 5-8 in: Proc. Cornell Nutr. Conf., Ithaca, NY.
Bloom, S. E., and L. D. Bacon, 1985. Linkage of the major histocompatibility (B) complex and the nucleolar organizer in the chicken: Assignment to a microchromosome. J. Hered. 76:146-154. Bloom, S. E., and E. G. Buss, 1969. Ammoniacal silver staining of embryonic chicken cells and chromosomes. Poultry Sci. 48:1114-1116. Bloom, S. E., P. Shalit, and L. D. Bacon, 1978. Chromosomal localization of nucleolus organizers in the chicken. Genetics 88:S13. (Abstr.) Bloom, S. E., E. M. Willand, and M. E. Delany, 1985. Development potential of chickens aneuploid for the rDNA/MHC microchromosome. Genetics 110:S3. (Abstr.) Briles, W. E., R. W. Briles, and R. E. Taffs, 1982. An apparent recombinant within the B-G region of the B complex. Poultry Sci. 61:1425-1426 Briles, W. E., and D. G. Gilmour, 1979. Blood group systems: Chicken. Part 1. Erythrocyte alloantigen characteristics. Page 650 in: Inbred and Genetically Defined Strains of Laboratory Animals. Part 2. P. L. Altman, and D. D. Katz, ed. Fed. Am. Soc. Exp. Biol. Handbook III. Briles, W. E., W. H. McGibbon, and M. R. Irwin, 1950. On multiple alleles effecting cellular antigens in the chicken. Genetics 35:633-652. Brown, W. V., 1972. Textbook of Cytogenetics. C. V. Mosby Co., St. Louis, MO. Delany, M. E., W. E. Briles, R. W. Briles, R. R. Dietert, E. M. Willand, and S. E. Bloom, 1985. Cell surface expression of major histocompatibility complex-encoded glycoprotein antigens in normal and aneuploid chickens. Genetics 110:S27. (Abstr.) Delany, M. E., R. R. Dietert, and S. E. Bloom, 1986. Increased membrane expression of la antigen and altered cell populations in bursa of aneuploid chickens. Poultry Sci. 65(Suppl 1):33. (Abstr.) Etches, R. J., and R. O. Hawes, 1979. Linkage relationship: Chicken. Part IV. Genes demonstrating independent segregation. Pages 628-637 in: Inbred and Genetically Defined Strains of Laboratory Animals. P. L. Altman and D. D. Katz, ed. Fed. Am. Soc. Exp. Biol., Bethesda, MD. Ewert, D. L., M. S. Munchus, Chen-Lo H. Chen, and M. D. Cooper, 1984. Analysis of structural properties and cellular distribution of avian la antigen by using monoclonal antibody to monomorphic determinants. J. Immunol. 132:2524-2530. Goodpasture, C , and S. E. Bloom, 1975. Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining. Chromosoma 53:37-50. Hala, K., M. Vilhelmova, and J. Hartmanova, 1976. Probable crossing-over in the B blood group of chickens. Immunogenetics 3:97-104. Jinks-Robertson, S., R. L. Gourse, and M. Nomura, 1983. Expression of rRNA and tRNA genes in Escherichia coli: evidence for feedback regulation by products of rRNA operons. Cell 33:865-876. Klein, J., 1975. Biology of the Mouse Histocompatibility Complex. Springer Verlag, Berlin, BRD. Krider, H. M., and W. Plaut, 1972. Studies on nucleolar RNA synthesis in Drosophila melanogaster I: the relationship between number of nucleolar organizers and rate of synthesis. J. Cell Sci. 11:675-687. Macera, M. J., and S. E. Bloom, 1981. Ultrastructural studies of the nucleoli in diploid and trisomic chickens. J. Hered. 72:249-252.
Downloaded from http://ps.oxfordjournals.org/ at Boston University on June 27, 2015
The discovery of aneuploids for the MHC not only facilitated its mapping but also aided studies of gene regulation and gene dosage effects in development. The picture emerging is that of differential gene expression of MHC and rDNA on the chromosome. Although the rDNA is regulated on each chromosome, the MHC appears to be expressed and we find extra cell surface products on both erythrocytes and B lymphocytes. Thus, we do not see evidence for whole chromosome inactivation to achieve dosage regulation in aneuploid avian cells. These and other considerations support the use of aneuploid chickens as a model to examine MHC gene dosage effects on developing systems in the chicken. Studies with this and other systems, such as congenic lines, will allow the dissection of the range of biologic influence of the MHC as a gene cluster. Such studies have already implicated the MHC as one factor influencing hatchability, postnatal growth potential, and even reproductive performance. The MHC influence, therefore, apparently extends beyond its actions in regulating the immune response to other traits of economic importance. It may not be entirely unreasonable to speculate at this stage that the MHC is a major fitness complex for Gallus domesticus and other species. This effect on fitness, related to the activity of the MHC, is hypothesized to consist of alterations in cell differentiation and tissue formations. Finally, the chicken MHC is on the map. It now stands along side of the mouse H-2 complex on Chromosome 17 and the human HLA on Chromosome 6. It will be of great interest to determine the extent of sequence and chromosome homology among species and to learn of the most highly conserved vs. highly evolving MHC sequences and functions.
SYMPOSIUM: AVIAN MAJOR HISTOCOMPATIBILITY COMPLEX
A. Ziegler, 1977. A three-locus model for the chicken major histocompatibility complex. Immunogenetics 5:203-216. Qureshi, M. A., S. E. Bloom, and R. R. Dietert, 1986. Macrophage function in chickens having increased copy number. Poultry Science 65(Suppl. I): 108. (Abstr.) Sandnes, G. C , 1954. A new technique for the study of avian chromosomes. Science 119:508-509. Schierman, L. W., and A. W. Nordskog, 1961. Relationship of blood type to histocompatibility in chickens. Science 134:1008-1009 Swanson, C. P., 1960. Cytology and Cytogenetics, 3rd ed. Prentice Hall, Inc., Englewood Cliffs, NJ. Tantravahi, U., R. V. Guntaka, B. F. Erlanger, and O. J. Miller, 1981. Amplified ribosomal RNA genes in a rat hepatoma cell line are enriched in 5-methyIcytosine. Proc. Natl. Acad. Sci. 78:489^193. Tartof, K. D., 1974. Unequal meitotic sister chromatid exchange as the mechanism of ribosomal RNA gene magnification. Proc. Natl. Acad. Sci. 71:1272-1276. Van Someren, H., A. Westerveld, A. Hagemaijer, J. R. Mees, K. Meera, and O. B. Zaalberg, 1974. Human antigen and enzyme markers in man-Chinese hamster somatic cell hybrids: evidence for synteny between the HL-A, PGMe, ME1, and IPO-B loci. Proc. Natl. Acad. Sci. 71:962-965.
Downloaded from http://ps.oxfordjournals.org/ at Boston University on June 27, 2015
Miller, M. M., Goto, R., and W. E. Briles, 1986. Biochemical evidence for recombination within the B-G region of the chicken major histocompatibility complex. Poultry Sci. 65(Suppl. 1):94. (Abstr.) Miller, O. J., D. A. Miller, V. G. Dev, R. Tantravahi, and C M . Croce, 1976. Expression of human and suppression of mouse nucleolus organizer activity in mousehuman somatic cell hybrids. Proc. Natl. Acad. Sci. 73:4531-4535. Muscarella, D. E., V. M. Vogt, and S. E. Bloom, 1985. The ribosomal RNA gene cluster in aneuploid chickens: evidence for increased gene dosage and regulation of gene expression. J. Cell Biol. 101:1749-1756. Nomura, M. R., L. Gourse, andG. Baughman, 1984. Regulation of the synthesis of ribosomes and ribosomal components. Annu. Rev. Biochem. 53:75-117. Ohno, S., 1961. Sex chromosomes and microchromosomes of Gallus domesticus. Chromosoma 11:484-498. Owen, J.J.T., 1965. Karyotype studies on Gallus domesticus. Chromosoma 16:601-608. Pazderka, F., B. M. Longenecker, G.R.J. Law, and R. F. Ruth, 1975. The major histocompatibility complex of the chicken. Immunogenetics 2:101-130. Petes, T. D., 1980. Unequal miotic recombination within tandem arrays of yeast ribosomal DNA genes. Cell 19:765-774. Pink, J.R.L., W. Droege, K. Hala, V. C. Miggiano, and
789