Fc gamma receptors differ in their structural requirements for interaction with the tyrosine kinase Syk in the initial steps of signaling for phagocytosis.

Receptors for the constant region of IgG, Fc gamma receptors, are expressed on the surface of hematopoietic cells, where they mediate signaling events, such as phagocytosis, essential for host defense. Fc gamma receptors also play a role in the pathophysiology of autoimmune diseases. We have demonstrated that members of each of the three classes of human Fc gamma receptors, Fc gamma RI, Fc gamma RII, and Fc gamma RIII, mediate phagocytosis, but that important differences exist in their requirements for phagocytic signaling. For example, the Fc gamma receptors Fc gamma RI and Fc gamma RIIIA induce signaling largely by association with a gamma subunit containing a conserved cytoplasmic motif (ITAM) whose tyrosines are phosphorylated following receptor stimulation. Fc gamma RIIA contains a similar motif in its own cytoplasmic domain and does not require the gamma chain for phagocytic signaling. The tyrosine kinase Syk associates with the cytoplasmic domain of both the Fc gamma receptor gamma chain and Fc gamma RIIA and is required for phagocytosis by both Fc gamma receptor systems. To elucidate the differences in phagocytic signaling by the gamma chain and Fc gamma RIIA, we investigated the requirements for Fc gamma receptor/Syk co-immunoprecipitation, tyrosine phosphorylation, and phagocytosis. Both Fc gamma RIIA and the human gamma chain contain a tyrosine seven amino acids upstream of the ITAM motif. We observed that the upstream tyrosine plays a role in Fc gamma RIIA phagocytic signaling but is not involved in phagocytic signaling by the human gamma chain. Our data also indicate that the two ITAM tyrosines of the human gamma chain and Fc gamma RIIA do not contribute equally to Fc gamma receptor association with Syk kinase and phagocytic signaling. The data indicate that the carboxy-terminal tyrosine of the receptor cytoplasmic domain is especially important both for the interaction with Syk kinase and for phagocytosis. Elucidating such differences in gamma chain and Fc gamma RIIA signaling may be valuable in designing strategies for therapeutic intervention in hematopoietic and immunological disorders.

The cytoplasmic domain of human FcgammaRIa alters the functional properties of the FcgammaRI.gamma-chain receptor complex.

The gamma/zeta-chain family of proteins mediate cell activation for multiple immunoglobulin receptors. However, the recognition that these receptors may have distinct biologic functions suggests that additional signaling elements may contribute to functional diversity. We hypothesized that the cytoplasmic domain (CY) of the ligand binding alpha-chain alters the biological properties of the receptor complex. Using macrophage FcgammaRIa as a model system, we created stable transfectants expressing a full-length or a CY deletion mutant of human FcgammaRIa. Both receptors functionally associate with the endogenous murine gamma-chain. However, we have established that the CY of FcgammaRIa directly contributes to the functional properties of the receptor complex. Deletion of the FcgammaRIa CY leads to slower kinetics of receptor-specific phagocytosis and endocytosis as well as lower total phagocytosis despite identical levels of receptor expression. Deletion of the CY also converts the phenotype of calcium independent FcgammaRIa-specific phagocytosis to a calcium-dependent phenotype. Finally, deletion of the CY abrogates FcgammaRIa-specific secretion of interleukin-6 but does not affect production of interleukin-1beta. These results demonstrate a functional role for the CY of FcgammaRIa and provide a general model for understanding how multiple receptors that utilize the gamma-chain can generate diversity in function.

Activation of three classes of nonreceptor tyrosine kinases following Fc gamma receptor crosslinking in human monocytes.

Fc gamma receptors on monocytes/macrophages play an important role in both host defense and autoimmune disorders. Fc gamma receptor signaling can lead to such downstream events as phagocytosis and the release of intracellular cytokines and reactive oxygen species. Freshly isolated human monocytes express two major classes of Fc gamma receptor proteins, Fc gamma RI (CD64) and Fc gamma RII (CD32). Crosslinking of Fc gamma RI and Fc gamma RII gives rise to rapid and transient phosphorylation of multiple monocyte intracellular proteins including proteins of 40, 68-72, 75-85, 95, and 115-165 kDa. A 72-kDa protein was earlier identified as the tyrosine kinase Syk. Here we identify one of the proteins in the 115- to 165-kDa cluster as FAK, a protein tyrosine kinase localized to focal adhesions. A 68-kDa phosphoprotein was identified as paxillin, a cytoskeleton associated substrate for tyrosine kinases, and a 95-kDa protein was found to be the proto-oncogene product Vav. The Src family protein tyrosine kinase Fgr (p58) also displayed enhanced tyrosine phosphorylation after Fc gamma RI and Fc gamma RII crosslinking. Although Fc gamma RIIA utilizes tyrosines within its own cytoplasmic domain for signaling while Fc gamma RI utilizes the cytoplasmic tyrosines of its associated gamma subunit, our results indicate sharing of several proteins for signaling in monocytes by these Fc receptors. These molecules include three distinct classes of tyrosine kinases, Syk, FAK, and Fgr, and the functionally diverse proteins Vav and paxillin.

Inhibition of Fcgamma receptor-mediated phagocytosis by a nonphagocytic Fcgamma receptor.

There are three major classes of human Fcgamma receptors (FcgammaRI, FcgammaRII, and FcgammaRIII) and various isoforms of each class are capable of mediating phagocytosis. FcgammaRIIA is an unusual Fcgamma receptor in that it transmits a phagocytic signal in the absence of an additional receptor subunit. The cytoplasmic domain of FcgammaRIIA contains a conserved motif containing two copies of the sequence YXXL. The tyrosines (Y) within the motif are phosphorylated after receptor crosslinking and the integrity of these conserved sequences is required for efficient phagocytosis. The FcgammaRIIB receptors, FcgammaRIIB1 and FcgammaRIIB2, contain one copy of the cytoplasmic YXXL sequence and do not transmit a phagocytic signal. In B cells, FcgammaRIIB negatively regulates B-cell activation by the B-cell antigen receptor. Human macrophages express both FcgammaRIIA and FcgammaRIIB and while FcgammaRIIA mediates phagocytosis, the function of FcgammaRIIB in these cells is unknown. Using the epithelial/fibroblast-like cell line COS-1 as a model to examine the molecular events that regulate the phagocytosis of IgG-coated cells (EA), we investigated the effect of FcgammaRIIB on FcgammaRIIA signaling. FcgammaRIIB inhibited phagocytosis mediated both by FcgammaRIIA and by a chimeric FcgammaRIIA receptor containing the extracellular domain of FcgammaRI and the transmembrane and cytoplasmic domains of FcgammaRIIA. This inhibition occurred at an early signaling stage because tyrosine phosphorylation of the FcgammaRIIA cytoplasmic domain was inhibited after concurrent stimulation of these receptors with EA. FcgammaRIIB mutations showed the importance of the FcgammaRIIB YXXL for inhibition of FcgammaRIIA-mediated phagocytosis. Deletion of the FcgammaRIIB YXXL or conservative replacement of the YXXL tyrosine substantially reduced the inhibitory signal. FcgammaRIIB had a lesser inhibitory effect on phagocytosis by the Fcgamma receptor FcgammaRIIIA, which requires a gamma subunit to mediate a phagocytic signal. These results show that FcgammaRIIB negatively regulates phagocytic signaling by FcgammaRIIA and suggests that FcgammaRIIB plays a role in modulating FcgammaRIIA function in vivo.

The molecular dissection of Fc gamma receptor mediated phagocytosis.

Because hematopoietic cells express multiple Fc gamma receptor isoforms, the role of the individual Fc gamma receptors in phagocytosis has been difficult to define. Transfection of Fc gamma receptors into COS-1 cells, which lack endogeneous Fc gamma receptors but have phagocytic potential, has proved valuable for the study of individual Fc gamma receptor function. Using this model system, we have established that a single class of human Fc gamma receptor mediates phagocytosis in the absence of other Fc receptors and that isoforms from each Fc gamma receptor class mediate phagocytosis, although the requirements for phagocytosis differ. In investigating the relationship between structure and function for Fc gamma receptor mediated phagocytosis, the importance of the cytoplasmic tyrosines of the receptor or its associated gamma chain has been established. For example, two cytoplasmic YXXL sequences, in a configuration similar to the conserved tyrosine-containing motif found in Ig gene family receptors, are important for phagocytosis by the human Fc gamma receptor, Fc gamma RIIA. Fc gamma RI and Fc gamma RIIIA do not possess cytoplasmic tyrosines but transmit a phagocytic signal through interaction with an associated gamma subunit that contains two YXXL sequences in a conserved motif required for phagocytosis. The human Fc gamma RII isoforms Fc gamma RIIB1 and Fc gamma RIIB2 do not induce phagocytosis and have only a single YXXL sequence. Cross-linking the phagocytic Fc gamma receptors induces tyrosine phosphorylation of either Fc gamma RIIA or the gamma chain, and treatment with tyrosine kinase inhibitors reduces both phagocytosis and phosphorylation of the receptor tyrosine residues. Activation of protein tyrosine kinases follows Fc gamma receptor engagement of IgG-coated cells. The data indicate that coexpression of the protein tyrosine kinase Syk, which is associated with the gamma chain in monocytes/macrophages, is important for phagocytosis mediated by Fc gamma RI and Fc gamma RIIIA. Furthermore, phosphatidylinositol-3 kinase is required for phagocytosis mediated by Fc gamma RIIA as well as for phagocytosis mediated by Fc gamma RI/gamma and Rc gamma RIIIA/gamma.

Induction of phagocytosis by a protein tyrosine kinase.

The transmission of extracellular signals to cellular targets by many noncatalytic surface receptors is dependent on interaction between cytoplasmic protein tyrosine kinases (PTKs) and tyrosine-containing sequences in the cytoplasmic domain of the receptor or an associated subunit. Isoforms of each of the three classes of the noncatalytic Fc gamma receptors, Fc gamma RI, Fc gamma RII, and Fc gamma RIII, are able to transmit a phagocytic signal in transfected COS-1 cells. Both Fc gamma RI and Fc gamma RIIIA require the gamma subunit for this signaling event. The protein tyrosine kinase Syk dramatically enhances phagocytosis mediated by both these receptors and increases the number of cells able to mediate phagocytosis. Two gamma chain cytoplasmic YXXL sequences are required for this effect. The action of Syk is less pronounced on the phagocytic Fc gamma RII receptor, Fc gamma RIIA, which does not require the gamma chain for phagocytosis. However, Syk allows phagocytosis by the nonphagocytic Fc gamma RII receptor Fc gamma RIIB2, which contains only a single YXXL sequence, when an additional tyrosine-containing sequence, YMTL, is introduced. These studies indicate that the efficiency of phagocytosis is markedly enhanced by the presence of a specific protein tyrosine kinase.

Structure/function relationships of Fc gamma receptors in phagocytosis.

An important function of Fc gamma receptors is the ingestion or phagocytosis of IgG sensitized cells. It has been difficult to clearly define the individual function of each receptor in phagocytosis because hematopoietic cells express multiple Fc gamma receptor isoforms. To examine this issue, an in-vitro model system in COS-1 cells has been developed. When transfected with an appropriate Fc gamma receptor, COS-1 cells which lack endogeneous Fc receptors, ingest IgG-sensitized cells. Using this model, a single class of human Fc gamma receptor in the absence of other Fc receptors was observed to mediate phagocytosis. Furthermore, isoforms from each Fc gamma receptor class can mediate phagocytosis although the requirements for phagocytosis differ. Investigation of the relationship between structure and function for Fc receptor-mediated phagocytosis established the importance of the cytoplasmic tyrosines of the receptor or its associated gamma chains. For example, two cytoplasmic YXXL sequences, in a configuration similar to the conserved tyrosine containing motif found in immunoglobulin gene family receptors, are important for phagocytosis by the human Fc gamma receptor, Fc gamma RIIA. Fc gamma RI and Fc gamma RIIIA do not possess cytoplasmic tyrosines, but transmit a phagocytic signal through interaction with an associated gamma-subunit which contains two YXXL sequences in a conserved motif required for phagocytosis. The human Fc gamma RII isoforms, Fc gamma RIIB2, do not induce phagocytosis and have only a single YXXL sequence. Crosslinking of the phagocytic Fc gamma receptors induces tyrosine phosphorylation of either Fc gamma RIIA or the gamma chain and treatment with tyrosine kinase inhibitors reduces both phagocytosis and phosphorylation of the receptor tyrosine residues. The protein tyrosine kinase Syk, which is associated with the gamma chain in monocytes/macrophages, dramatically enhances phagocytosis mediated by Fc gamma RI and Fc gamma RIIIA and also induces non-phagocytic Fc gamma RI or Fc gamma RIIIA expressing cells to acquire phagocytic capability.

Molecular dissection of Fc gamma receptor-mediated phagocytosis.

Using an experimental model in COS-1 cells, we have examined the structural requirements for phagocytosis of IgG-sensitized cells by Fc gamma receptors. We have established that isoforms of each of the 3 classes of the Fc gamma receptors, Fc gamma RI, Fc gamma RII and Fc gamma RIII, are able to transmit a phagocytic signal in the absence of the other receptor class. Fc gamma I and Rc gamma RIIIA require a gamma-subunit for this signaling event, but Fc gamma RIIA does not. Fc gamma RIIA and the gamma-subunit associated with Fc gamma RI and Fc gamma RIIIA contain 2 copies of a conserved tyrosine-containing cytoplasmic sequence, YXXL. This sequence is important for phagocytosis and is phosphorylated on tyrosine after receptor ligation. The Fc gamma receptors Fc gamma RIIB1 and Fc gamma RIIB2 which contain only 1 copy of the YXXL cytoplasmic sequence do not include the phagocytosis of IgG-coated cells. Thus, the Fc gamma receptor isoforms differ in their ability to transmit a phagocytic signal. Structure/function studies also indicate that the Fc gamma receptors which induce phagocytosis differ in their requirements for phagocytic signaling.

Substitutions and deletions in the cytoplasmic domain of the phagocytic receptor Fc gamma RIIA: effect on receptor tyrosine phosphorylation and phagocytosis.

Fc gamma RIIA in the absence of other Fc receptors or receptor subunits induces the ingestion of IgG-coated cells. The cytoplasmic domain of Fc gamma RIIA contains two Y-x-x-L sequences similar to those in other Ig gene family receptors plus an additional tyrosine residue not in a Y-x-x-L motif. Upon cross-linking, Fc gamma RIIA is phosphorylated on tyrosine and the cytoplasmic tyrosines, Y275 (Y1), Y282 (Y2), and Y298 (Y3), may be important for its phagocytic activity. Because COS-1 cells can serve as a model for examining molecular structures involved in phagocytosis, substitutions and deletions were introduced into the cytoplasmic domain of Fc gamma RIIA and examined in COS-1 cell transfectants for their effects on phagocytosis and tyrosine phosphorylation. Disruption of a single cytoplasmic Y-x-x-L motif by substitution of tyrosine Y2 or Y3 by phenylalanine or by removing the threonine and leucine residues within the motif inhibited phagocytosis 50% to 65%. Tyrosine phosphorylation of Fc gamma RIIA also was inhibited, although to a greater extent by the substitution of Y3 than of Y2. Replacement of the N-terminal first cytoplasmic domain tyrosine, Y1, which is not within a typical Y-x-x-L, by itself did not inhibit phagocytosis, but replacement of Y1 in mutants lacking Y2 or Y3 virtually eliminated phagocytic activity and receptor tyrosine phosphorylation. Thus, at least two cytoplasmic tyrosines, including at least one typical single Y-x-x-L motif, are required for phagocytosis by Fc gamma RIIA. The data suggest that there is a close but not a simple relationship between phosphorylation of the Fc gamma RIIA cytoplasmic tyrosines and Fc gamma RIIA-mediated phagocytosis. Y3 appears to be particularly important because its removal by truncation or replacement with phenylalanine inhibits both tyrosine phosphorylation and phagocytosis in parallel. Alterations in the 12 residue proline-containing sequence between the two Y-x-x-L motifs also reduced phagocytic activity and tyrosine phosphorylation. Thus, the specific structure of the Fc gamma RIIA cytoplasmic domain accounts for its ability to stimulate phagocytosis in the absence of other subunits.

The high affinity Fc gamma receptor (CD64) induces phagocytosis in the absence of its cytoplasmic domain: the gamma subunit of Fc gamma RIIIA imparts phagocytic function to Fc gamma RI.

The high affinity Fc gamma receptor, Fc gamma RI, is unique among the three classes of macrophage Fc gamma receptors not only in its affinity for IgG, but also in the structure of its cytoplasmic domain. Fc gamma RIIA and the gamma subunit of Fc gamma RIIIA have tyrosine-containing motifs within their cytoplasmic domains that are phosphorylated when crosslinked and that are required for phagocytosis by COS-1 cell transfectants. In contrast to these other Fc gamma receptors, Fc gamma RI does not contain cytoplasmic tyrosines and does not induce phagocytosis in COS-1 transfectants. We transfected wild-type (WT) and mutant (MT) Fc gamma RI lacking the cytoplasmic domain into COS-1 cells and murine macrophages and assessed phagocytosis using IgG-coated red blood cells (RBCs) and RBCs conjugated with Fab anti-human Fc gamma RI monoclonal antibody (mAb). Fc gamma RI, in contrast to Fc gamma RIIA, did not induce phagocytosis in COS cells. However, both WT and MT Fc gamma RI induced phagocytosis in murine macrophages, and phagocytosis was inhibited by the tyrosine kinase inhibitor tyrphostin 23. Human monocytes also phagocytosed Fc gamma RI-targeted RBCs, and activation of Fc gamma RI on monocytes with Fab anti-Fc gamma RI induced phosphorylation of Fc gamma RII on tyrosine residues. However, Fc gamma RI activation of Fc gamma RI-Fc gamma RIIA COS-1 cotransfectants did not induce tyrosine phosphorylation of Fc gamma RIIA, and coexpression of Fc gamma RI and Fc gamma RIIA in COS cells did not confer Fc gamma RI phagocytic capability. In contrast, coexpression in COS-1 cells of Fc gamma RI with the gamma subunit of Fc gamma RIIIA conferred phagocytic function to both Fc gamma RI and the MT Fc gamma RI lacking the cytoplasmic domain. Thus, Fc gamma RI does not require its cytoplasmic domain to mediate a phagocytic signal and interacts with the gamma subunit of Fc gamma RIIIA to induce phagocytosis.

Insertion of cytoplasmic tyrosine sequences into the nonphagocytic receptor Fc gamma RIIB establishes phagocytic function.

Receptors for the Fc domain of IgG on cells of hematopoietic lineage perform important functions, including stimulation of the ingestion of IgG-coated cells. In examining the function of Fc gamma receptor isoforms by transfection into COS-1 cells, we have observed that Fc gamma RIIA induces the binding and phagocytosis of IgG-sensitized RBCs (EA) and that transfected COS-1 cells can serve as a model for examining the molecular structures involved in mediating a phagocytic signal. We now report that COS-1 cell transfectants expressing the isoforms Fc gamma RIIB1 and Fc gamma RIIB2 and a Fc gamma RIIA mutant without a cytoplasmic tail efficiently bind EA but do not mediate their phagocytosis. Furthermore, wild-type Fc gamma RIIA, but not Fc gamma RIIB1 or Fc gamma RBII2, was phosphorylated on tyrosine upon receptor activation. Tyrphostin 23, which alters tyrosine kinase activity, inhibited the phagocytosis of EA and reduced the phosphorylation of Fc gamma RIIA on tyrosine. Fc gamma RIIB1 and Fc gamma RIIB2 contain one copy of the cytoplasmic sequence YXXL/I implicated in signal transduction, whereas Fc gamma RIIA contains two copies. We therefore inserted YXXL/I sequences at different sites in Fc gamma RIIB2. Low levels of phagocytosis were observed in a Fc gamma RIIB2 mutant bearing the Fc gamma RIIA sequence YMTL and higher levels of phagocytosis were observed in a second Fc gamma RIIB2 mutant that contained both the upstream YMTL and an additional downstream tyrosine-containing motif. Activation of this mutant receptor also induced receptor tyrosine phosphorylation. Thus, these studies indicate that both the number and placement of YXXL sequences in the cytoplasmic domain of the Fc gamma RII receptor family affect both receptor tyrosine phosphorylation and phagocytic competence.

Fc gamma RIIA-mediated phagocytosis and receptor phosphorylation in cells deficient in the protein tyrosine kinase Src.

In the absence of other Fc receptors, stimulation of Fc gamma RIIA induces receptor phosphorylation and phagocytosis of immunoglobulin G (IgG)-coated cells. In vitro, Fc gamma RIIA is phosphorylated by the Src-related tyrosine kinase (SRTK) Src. Therefore, we investigated whether fibroblasts transfected with Fc gamma RIIA mediate phagocytosis of IgG-coated cells and whether Src is required for Fc gamma RIIA phosphorylation and for phagocytosis in vivo. Activation of Fc gamma RIIA in a fibroblast cell line deficient in Src kinase resulted in phosphorylation of the receptor on tyrosine. In addition, Fc gamma RIIA-mediated phagocytosis was observed in these fibroblasts in both the presence and absence of Src. In the presence of Src, however, phagocytosis of IgG-coated cells was more efficient. The data indicate that the SRTK Src is not required for Fc gamma RIIA phosphorylation or for Fc gamma RIIA-mediated phagocytosis in these cells. In vitro kinase assays demonstrated that the SRTK Fyn also is able to phosphorylate Fc gamma RIIA. Thus, Fc gamma RIIA can be phosphorylated by more than one tyrosine kinase in vitro. The data suggest that there may be shared functions among some intracellular kinases in receptor phosphorylation.

Chromosomal localization of the human elastin gene.

mRNA isolated from fetal human aorta was used to synthesize cDNA that was cloned into the PstI site of pBR322. The recombinant clones were screened with an authentic sheep elastin cDNA, and one human clone that hybridized strongly was isolated and characterized. The 421-base pair (bp) insert of this human clone was sequenced by the dideoxy method, and the DNA sequence showed strong homology to the nontranslated portion of the sheep elastin cDNA. This result unequivocally identified the human clone, designated pcHEL1, as an elastin clone. Plasmid pcHEL1 labeled with [3H] nucleotides was used in in situ hybridization experiments utilizing normal metaphase chromosomes and also with cells carrying a balanced translocation between chromosomes 1 and 2: 46,XY,t(1;2)(p36;q31). The results strongly suggest that the elastin gene is localized to the q31----qter region of chromosome 2.

Characterization of a sheep elastin cDNA clone containing translated sequences.

mRNA, isolated from the ligamentum nuchae of fetal sheep by guanidine HCl extraction and oligo(dT) cellulose chromatography, was used to synthesize blunt-ended cDNA molecules by the successive application of AMV reverse transcriptase, DNA polymerase and S1 nuclease. The cDNA was centrifuged on a 15-30% sucrose gradient and molecules greater than 700 bp were tailed with dCTP and cloned into the PstI site of pBR322 which had been tailed with dGTP. Ampicillin-sensitive and tetracycline-resistant colonies were screened by in situ hybridization with elastin-enriched mRNA that had been terminally labeled with 32p. Recombinant plasmids prepared from strongly hybridizing colonies were characterized by restriction mapping and the plasmid with the largest insert (1300 bp) thought to contain elastin sequences was characterized in more detail. The nick-translated cDNA hybridized to a single 3.5 kb mRNA species upon blot hybridization, a size identical to that previously identified for chick elastin mRNA (Burnett et al. (1982) J. Biol. Chem. 259, 1569-1572). Nucleotide sequencing of the 5' end of the cDNA demonstrated a sequence which was extremely GC rich and which corresponded to an amino acid sequence partially homologous to that previously identified in porcine tropoelastin (Foster et al. (1973) J. Biol. Chem. 248, 2876-2879). This is the first report of the identification of a plasmid containing sequences complementary to a translated region of elastin mRNA.

Glutamate tRNA genes are adjacent to 5S RNA genes in Drosophila and reveal a conserved upstream sequence (the ACT-TA box).

In Drosophila melanogaster at least six transfer RNA genes are located adjacent to the 3' end of the 5S RNA gene cluster. Three of these have been sequenced and identified as coding for glutamate tRNA4. In the chromosome they are arranged as tandem repeats on the same DNA strand and transcribed in the same direction as is 5S DNA, towards the centromere. We have also identified a sequence, the ACT-TA box, that is highly conserved among the polymerase III transcribed genes. Usually the sequence is located at 37 +/- 8 base pairs upstream from the first nucleotide of the structural gene. A similar sequence is also observed upstream of yeast and silkworm tRNA genes and the mitochondrial tRNA genes of mouse and humans.

Long spacers among ribosomal genes of Drosophila melanogaster.

The fruit fly, Drosophila melanogaster, has 200 tandemly arranged copies of the ribosomal RNA genes (rDNA) per haploid genome. One such cluster of rRNA genes occurs on the X and one on the Y chromosome. The basic repeating unit of the rRNA gene consists of a segment coding for 18S rRNA and 28S rRNA followed by a non-transcribed spacer (Fig.1). In the X chromosome, there are two major size classes (12 and 17 kilobases) and numerous minor size classes of rDNA repeats. Most of this length heterogeneity is generated by insertions at a specific site in the 28S gene. The frequency and size patterns of these insertions in the 28S gene differ in the X and Y chromosomes. Electron microscopic analysis of rDNA-rRNA hybrids has shown that there is also length heterogeneity in the rDNA non-transcribed spacer. This heterogeneity is due in part to internal sequence repetition. We have now examined further the length heterogeneity of the rDNA spacer and have observed a class of spacers that we shall refer to as 'long spacers'. The size and frequency of these long spacers are different in the X and Y rDNA.