Analysis of nuclear lamin isoprenylation in Xenopus oocytes: isoprenylation of lamin B3 precedes its uptake into the nucleus.

Protein prenylation is a posttranslational modification involving the covalent attachment of a prenyl lipid to a cysteine at or near the COOH terminus of a protein. It is required for membrane localization and efficient function of a number of cytoplasmic as well as nuclear proteins including the proto-oncogenic and activated forms of Ras. Farnesylation in conjunction with a nuclear localization signal has been shown to be necessary to target newly synthesized nuclear lamins to the inner nuclear envelope membrane. It is, however, not clear where in the cell isoprenylation of nuclear lamins takes place. In this study we describe in vivo and in vitro experiments on the isoprenylation of the Xenopus oocyte nuclear lamin B3. We show by kinetic analysis that newly synthesized lamins are isoprenylated in the cytosol of oocytes before uptake into the nucleus. From our data it can be concluded that isoprenylation of lamins in the nucleus, as it is observed under certain conditions of isoprene starvation, represents a default pathway rather than the physiological situation. We further analyzed the capacity of isolated nuclei to carry out isoprenylation of B3. Our results are in line with a dual localization of a protein farnesyltransferase in the cytosol and nuclei of amphibian oocytes. Implications for the possible functions of a nuclear protein farnesyltransferase as well as possible mechanisms of the selective inhibition of farnesylation of cytoplasmic proteins by peptidomimetics are discussed.

The role of CaaX-dependent modifications in membrane association of Xenopus nuclear lamin B3 during meiosis and the fate of B3 in transfected mitotic cells.

Recent evidence shows that the COOH-terminal CaaX motif of lamins is necessary to target newly synthesized proteins to the nuclear envelope membranes. Isoprenylation at the CaaX-cysteine has been taken to explain the different fates of A- and B-type lamins during cell division. A-type lamins, which loose their isoprenylation shortly after incorporation into the lamina structure, become freely soluble upon mitotic nuclear envelope breakdown. Somatic B-type lamins, in contrast, are permanently isoprenylated and, although depolymerized during mitosis, remain associated with remnants of nuclear envelope membranes. However, Xenopus lamin B3, the major B-type lamin of amphibian oocytes and eggs, becomes soluble after nuclear envelope breakdown in meiotic metaphase. Here we show that Xenopus lamin B3 is permanently isoprenylated and carboxyl methylated in oocytes (interphase) and eggs (meiotic metaphase). When transfected into mouse L cells Xenopus lamin B3 is integrated into the host lamina and responds to cell cycle signals in a normal fashion. Notably, the ectopically expressed Xenopus lamin does not form heterooligomers with the endogenous lamins as revealed by a coprecipitation experiment with mitotic lamins. In contrast to the situation in amphibian eggs, a significant portion of lamin B3 remains associated with membranes during mitosis. We conclude from these data that the CaaX motif-mediated modifications, although necessary, are not sufficient for a stable association of lamins with membranes and that additional factors are involved in lamin-membrane binding.

The fates of chicken nuclear lamin proteins during mitosis: evidence for a reversible redistribution of lamin B2 between inner nuclear membrane and elements of the endoplasmic reticulum.

In chicken, three structurally distinct nuclear lamin proteins have been described. According to their migration on two-dimensional gels, these proteins have been designated as lamins A, B1, and B2. To investigate the functional relationship between chicken lamins and their mammalian counterparts, we have examined here the state of individual chicken lamin proteins during mitosis. Current models proposing functional specializations of mammalian lamin subtypes are in fact largely based on the observation that during mitosis mammalian lamin B remains associated with membrane vesicles, whereas lamins A and C become freely soluble. Cell fractionation experiments combined with immunoblotting show that during mitosis both chicken lamins B1 and B2 remain associated with membranes, whereas lamin A exists in a soluble form. In situ immunoelectron microscopy carried out on mitotic cells also reveals membrane association of lamin B2, whereas the distribution of lamin A is random. From these results we conclude that both chicken lamins B1 and B2 may functionally resemble mammalian lamin B. Interestingly, immunolabeling of mitotic cells revealed an association of lamin B2 with extended membrane cisternae that resembled elements of the endoplasmic reticulum. Quantitatively, we found that all large endoplasmic reticulum-like membranes present in metaphase cells were decorated with lamin B2-specific antibodies. Given that labeling of these mitotic membranes was lower than labeling of interphase nuclear envelopes, it appears likely that during mitotic disassembly and reassembly of the nuclear envelope lamin B2 may reversibly distribute between the inner nuclear membrane and the endoplasmic reticulum.

Disruption of nuclear lamin organization alters the distribution of replication factors and inhibits DNA synthesis.

The nuclear lamina is a fibrous structure that lies at the interface between the nuclear envelope and the nucleoplasm. The major proteins comprising the lamina, the nuclear lamins, are also found in foci in the nucleoplasm, distinct from the peripheral lamina. The nuclear lamins have been associated with a number of processes in the nucleus, including DNA replication. To further characterize the specific role of lamins in DNA replication, we have used a truncated human lamin as a dominant negative mutant to perturb lamin organization. This protein disrupts the lamin organization of nuclei when microinjected into mammalian cells and also disrupts the lamin organization of in vitro assembled nuclei when added to Xenopus laevis interphase egg extracts. In both cases, the lamina appears to be completely absent, and instead the endogenous lamins and the mutant lamin protein are found in nucleoplasmic aggregates. Coincident with the disruption of lamin organization, there is a dramatic reduction in DNA replication. As a consequence of this disruption, the distributions of PCNA and the large subunit of the RFC complex, proteins required for the elongation phase of DNA replication, are altered such that they are found within the intranucleoplasmic lamin aggregates. In contrast, the distribution of XMCM3, XORC2, and DNA polymerase alpha, proteins required for the initiation stage of DNA replication, remains unaltered. The data presented demonstrate that the nuclear lamins may be required for the elongation phase of DNA replication.

Differential expression of nuclear lamin proteins during chicken development.

By immunocytochemistry, quantitative immunoblotting, and two-dimensional gel electrophoresis, we have analyzed the distribution of nuclear lamin proteins during chicken embryonic development. Whereas no qualitative differences in the patterns of expression of lamins A, B1, and B2 were observed during gametogenesis in either the female or the male germ line, profound changes in the composition of the nuclear lamina occurred during the development of somatic tissues. Most unexpectedly, early chicken embryos were found to contain little if any lamin A, although they contained substantial amounts of lamins B1 and B2. During embryonic development, lamin A became increasingly prominent, whereas the amounts of lamin B1 decreased in many tissues. Interestingly, the extent and the developmental timing of these changes displayed pronounced tissue-specific variations. Lamin B2 was expressed in fairly constant amounts in all cell types investigated (except for pachytene-stage germ cells). These results have implications for the purported functional specializations of individual lamin proteins. In addition, they suggest that alterations in the composition of the nuclear lamina may be important for the establishment of cell- or tissue-specific differences in nuclear architecture.

Major epiplasmic proteins of ciliates are articulins: cloning, recombinant expression, and structural characterization.

The cytoskeleton of certain protists comprises an extensive membrane skeleton, the epiplasm, which contributes to the cell shape and patterning of the species-specific cortical architecture. The isolated epiplasm of the ciliated protist Pseudomicrothorax dubius consists of two major groups of proteins with molecular masses of 78-80 kD and 11-13 kD, respectively. To characterize the structure of these proteins, peptide sequences of two major polypeptides (78-80 kD) as well as a cDNA representing the entire coding sequence of a minor and hitherto unidentified component (60 kD; p60) of the epiplasm have been determined. All three polypeptides share sequence similarities. They contain repeated valine- and proline-rich motifs of 12 residues with the consensus VPVP--V-V-V-. In p60 the central core domain consists of 24 tandemly repeated VPV motifs. Within the repeat motifs positively and negatively charged residues, when present, show an alternating pattern in register with the V and P positions. Recombinant p60 was purified in 8 M urea and dialyzed against buffer. Infrared spectroscopic measurements indicate 30% beta-sheet. Electron microscopy reveals short filamentous polymers with a rather homogenous diameter (approximately 15-20 nm), but variable lengths. The small polymers form thicker filaments, ribbons, and larger sheets or tubes. A core domain similar to that of P. dubius p60 is also found in the recently described epiplasmic proteins of the flagellate Euglena, the so-called articulins. Our results show that the members of this protein family are not restricted to flagellates, but are also present in the distantly related ciliates where they are major constituents of the epiplasm. Comparison of flagellate and ciliate articulins highlights common features of this novel family of cytoskeletal proteins.

Immunological localization of a major karyoskeletal protein in nucleoli of oocytes and somatic cells of Xenopus laevis.

Oocyte nuclei of Xenopus laevis contain two major karyoskeletal proteins characterized by their resistance to extractions in high salt buffers and the detergent Triton X-100, i.e. a polypeptide of 68,000 mol wt which is located in the core complex-lamina structure and a polypeptide of 145,000 mol wt enriched in nucleolar fractions. Both proteins are also different by tryptic peptide maps and immunological determinants. Mouse antibodies were raised against insoluble karyoskeletal proteins from Xenopus oocytes and analyzed by immunoblotting procedures. Affinity purified antibodies were prepared using antigens bound to nitrocellulose paper. In immunofluorescence microscopy of Xenopus oocytes purified antibodies against the polypeptide of 145,000 mol wt showed strong staining of nucleoli, with higher concentration in the nucleolar cortex, and of smaller nucleoplasmic bodies. In various other cells including hepatocytes, Sertoli cells, spermatogonia, and cultured kidney epithelial cells antibody staining was localized in small subnucleolar granules. The results support the conclusion that this "insoluble" protein is a major nucleus-specific protein which is specifically associated with--and characteristic of--nucleoli and certain nucleolus-related nuclear bodies. It represents the first case of a positive localization of a karyoskeletal protein in the nuclear interior, i.e. away from the pore complex-lamina structure of the nuclear cortex.

A role for nuclear lamins in nuclear envelope assembly.

The molecular interactions responsible for nuclear envelope assembly after mitosis are not well understood. In this study, we demonstrate that a peptide consisting of the COOH-terminal domain of Xenopus lamin B3 (LB3T) prevents nuclear envelope assembly in Xenopus interphase extracts. Specifically, LB3T inhibits chromatin decondensation and blocks the formation of both the nuclear lamina-pore complex and nuclear membranes. Under these conditions, some vesicles bind to the peripheral regions of the chromatin. These "nonfusogenic" vesicles lack lamin B3 (LB3) and do not bind LB3T; however, "fusogenic" vesicles containing LB3 can bind LB3T, which blocks their association with chromatin and, subsequently, nuclear membrane assembly. LB3T also binds to chromatin in the absence of interphase extract, but only in the presence of purified LB3. Additionally, we show that LB3T inhibits normal lamin polymerization in vitro. These findings suggest that lamin polymerization is required for both chromatin decondensation and the binding of nuclear membrane precursors during the early stages of normal nuclear envelope assembly.

Association of prenylated proteins with the plasma membrane and the inner nuclear membrane is mediated by the same membrane-targeting motifs.

Targeting of nuclear lamins to the inner nuclear envelope membrane requires a nuclear localization signal and CaaX motif-dependent posttranslational modifications, including isoprenylation and carboxyl methylation. These modifications, although necessary for membrane targeting, are not sufficient to mediate stable association with membranes. We show that two variants of lamin B3 (i.e., B3a and B3b) exist in Xenopus oocytes. They are encoded by two alternatively spliced, developmentally regulated mRNAs. The two lamin variants differ greatly in their membrane association in meiotically matured eggs. The presence of an extra cysteine residue (as a potential palmitoylation site) and a basic cluster in conjunction with the CaaX motif function as secondary targeting signals responsible for the stable membrane association of lamin B3b in Xenopus eggs. Moreover, transfection experiments with Green Fluorescent Protein lamin tail chimeras and with a Green Fluorescent Protein N-Ras chimera show that these secondary motifs are sufficient to target proteins to the inner nuclear membrane and/or the plasma membrane. Implications for the intracellular trafficking of doubly lipidated proteins are discussed.

Plasma clearance of model lipoproteins containing saturated and polyunsaturated monoacylglycerols injected intravenously in the rat.

Triacylglycerols, with a saturated long-chain fatty acid at the glycerol-2-position, slow the clearance from plasma of remnants derived from injected chylomicrons and chylomicron-like emulsions. Slowing of remnant clearance also occurs when about 1% of monostearoylglycerol is added to a triolein chylomicron-like emulsion. We have now found that addition of monoacylglycerols, containing a saturated acyl chain from 12 to 20 carbons, slowed the plasma clearance and decreased the liver uptake of the remnants. In contrast, monoacylglycerols with unsaturated acyl chains were inconsistent in their effects on the remnant clearance. Monoarachidonin (M20:4) slowed remnant clearance comparable to that of saturated monoacylglycerols, monolinolenin (M18:3) and monolinolein (M18:2) were less effective, while monoolein had the least effect on remnant clearance. We have confirmed the defective remnant clearance in rats of injected emulsions containing saturated acyl chain by the using the diester-2-ether analogues of triolein and 1,3-dioleoyl-2-stearoylglycerol (OSO). Chylomicron-like lipid emulsions made with the ether analogues had clearance rates similar to their triester counterparts. Preformed remnants derived from emulsions of OSO, its ether analogue, and triolein emulsions or emulsions of triolein with approximately 1% saturated monoacylglycerols were prepared in hepatectomized rats. After intravenous injection into conscious recipient rats, these remnants were cleared from plasma similar to remnants traced in situ by lipolysis of injected chylomicron-like emulsions.

The effect of monostearoylglycerol on the metabolism of chylomicron-like lipid emulsions injected intravenously in rats.

In rats, remnant particles derived from chylomicron-like emulsions containing 1,3-dioleoyl-2-stearoylglycerol (OSO) are removed from plasma more slowly than remnants derived from triolein emulsions. The effect associated with a saturated acyl chain at the glycerol 2-position could be reproduced by incorporating 2-stearoylglycerol (MS) in a triolein emulsion. When MS solubilized with rat albumin or in plasma was injected before the injection of a triolein emulsion, clearance of the triolein emulsion was unchanged. The metabolic fate of MS, monitored with 14C-labelled MS, was similar whether incorporated in triacylglycerol emulsion or injected independently. More than 95% of MS had disappeared from the circulation by 5 min after the injection and the radioactivity was found in liver, spleen, muscle and adipose tissue. Some MS label appeared in plasma triacylglycerol. Remnants made in vitro by incubating triolein or OSO emulsions with post-heparin plasma showed no differences in their disappearance from plasma. With OSO emulsion, the in vitro remnants were found to contain more MS than remnants made in vivo in hepatectomized rats. Simultaneous injections of mixtures containing OSO and triolein emulsions, or triolein emulsions with and without MS, each labelled with either [3H]cholesteryl oleate or [14C]cholesteryl oleate showed consistently slower remnant removal and decreased liver uptake of the emulsions containing OSO or MS. Affinity columns and immunodiffusion all indicated that there was no difference in the amounts of apolipoprotein E associated with OSO or triolein particles. The protein spectra of in vivo remnants derived from OSO and triolein emulsion were also similar when examined by SDS-PAGE and isoelectric focusing gels. Our results show that the effects due to OSO or MS are mediated by the presence of MS in the emulsion particle surface, while indirect effects expressed in plasma or liver are excluded. The precise mechanism of the effect remains to be established, but it does not correlate with measurable changes in the spectra of apolipoproteins associated with the emulsion remnants.

The effect of added monoacylglycerols on the removal from plasma of chylomicron-like emulsions injected intravenously in rats.

Lipid emulsions were prepared with compositions similar to the triacylglycerol-rich plasma lipoproteins, but also incorporating added small amounts of monoacylglycerols. Control emulsions without monoacylglycerol were metabolized similarly to natural chylomicrons or very-low-density lipoproteins when injected intravenously in rats. The emulsion triacylglycerols and cholesteryl esters were both removed rapidly from the bloodstream, with the removal rates of triacylglycerols faster than those of cholesteryl esters. Much of the removed cholesteryl ester was found in the liver, but only a small fraction of the triacylglycerol, consistent with hepatic uptake of the triacylglycerol-depleted remnants of the injected emulsion. Emulsions incorporating added monooleoylglycerol or stearic acid were metabolized similarly. Added 1- or 2-monostearoylglycerol had no effect on triacylglycerol removal from plasma, but the removal rate of cholesteryl esters was decreased and less cholesteryl ester was found in the liver. These effects are similar to those recently described when emulsions and chylomicrons contained triacylglycerols with a saturated acyl chain at the glycerol 2-position, suggesting that saturated monoacylglycerol produced by the action of lipoprotein lipase may cause triacylglycerol-depleted remnant particles to remain in the plasma instead of being rapidly taken up by the liver.

Effects of triacylglycerol-saturated acyl chains on the clearance of chylomicron-like emulsions from the plasma of the rat.

We previously found that a single saturated acyl chain at the glycerol 2-position affected the metabolism of chylomicrons. The explanation for the effect is not clear, but could be reproduced by saturated monoacylglycerols. In the present work we have extended our measurements to several different triacylglycerols containing one or two saturated chains in specific locations in an attempt to define structural features that affect chylomicron clearance. Lipid emulsions containing triacylglycerol, egg yolk phosphatidylcholine, free cholesterol, cholesteryl oleate (CO) and labelled with 3H-CO and [14C]triolein (OOO) were prepared as models of lymph chylomicrons. When injected intravenously into rats, the metabolism of the emulsions was influenced by the acyl chains of the constituent triacylglycerols. Compared with emulsions containing OOO as the only triacylglycerol, plasma clearances of emulsion [3H]CO were extremely slow in emulsions containing either 1,2-dioleoyl-3-stearoylglycerol (OOS) or 1-stearoyl-2,3-dioleoylglycerol (SOO). As little as 10% of SOO in mixture with OOO slowed the clearance, and increasing proportions of SOO in OOO emulsions progressively slowed the removal of OOO and CO labels from plasma. With 50% and 100% SOO in the emulsions clearance was negligible. In emulsions containing the triacyl-sn-glycerols, 1,3-dimyristoyl-2-oleoylglycerol (MOM), 1,3-dipalmitoyl-2-oleoylglycerol (POP), 1-oleoyl-2,3-distearoylglycerol (OSS) or 1-palmitoyl-2-oleoyl-3-stearoylglycerol (POS), clearance rates of CO and OOO labels from plasma were significantly decreased compared with control OOO emulsions. With emulsions prepared with the triacylglycerols, 1-oleoyl-2,3-dimyristoylglycerol (OMM) and 1-oleoyl-2,3-dipalmitoylglycerol (OPP), clearances of CO label were significantly slower than with control OOO emulsions, while the removal of OOO label was not significantly affected. The uptake of CO label in the liver was decreased in conjunction with the lower rates of clearance of emulsion CO from the plasma. The clearance from plasma of 1,3-distearoyl-2-oleoylglycerol (SOS) emulsions was similar to the control OOO emulsions, but significantly more emulsion OOO label was taken up by the liver. Emulsions made with the triacylglycerols extracted from natural cocoa butter, which contained a high proportion of saturated acyl chains, were cleared similarly to the control OOO emulsions. Our findings indicate that the plasma clearance of triacylglycerol-rich lipoprotein particles depends upon the specific arrangements of the acyl chains of the constituent triacylglycerols, and not necessarily on the overall saturation of the triacylglycerols.(ABSTRACT TRUNCATED AT 400 WORDS)

Translational control of nuclear lamin B1 mRNA during oogenesis and early development of Xenopus.

Cytoplasmic polyadenylation of specific mRNAs is commonly correlated with their translational activation during development. A canonical nuclear polyadenylation element AAUAAA (NPE) and cytoplasmic polyadenylation element(s) (CPE) are necessary and sufficient for polyadenylation during egg maturation. We have characterized cis-acting sequences of Xenopus nuclear lamin B1 mRNA that mediate translational regulation. By injection of synthetic RNAs into oocytes we show that the two CPE-like elements found in the 3'-untranslated region of B1 mRNA act as translational repressors in oocytes. The same CPEs in conjunction with the NPE confer transient polyadenylation and translational activation during egg maturation. Poly(A) length determination of the endogenous lamin B1 mRNA reveals a gradual increase of poly(A) tail length in early development up to mid-blastula, and a shortening of poly(A) tails during gastrulation and neurulation. The same kinetic and extent of polyadenylation and poly(A) tail shortening is observed with synthetic RNAs injected into fertilized eggs. Polyadenylation and translational activation of these RNAs is independent of the two CPEs and a NPE during early development. While translational regulation of lamin B1 mRNA functions in parts via established mechanisms, the pattern of polyadenylation and deadenylation during early development points to a novel mode of translational regulation.

Xenopus Xsal-1, a vertebrate homolog of the region specific homeotic gene spalt of Drosophila.

We have isolated an amphibian homolog of the homeotic gene spalt of Drosophila. Like its Drosophila counterpart the Xenopus Xsal-1 gene encodes a protein that contains three widely separated sets of sequence related double zinc finger motifs of the CC/HH-type as well as a single CC/HH zinc finger. The Xenopus gene encodes a fourth double zinc finger and a single CC/HC zinc finger motif that have no counterpart in the fly protein. Alternative splicing of Xsal-1 transcripts gives rise to RNAs coding for either four, three or two double zinc fingers, respectively. The main expression domains of Xsal-1 in early development are confined to distinct regions along the lateral axon tracts within the midbrain, hindbrain, and spinal cord. Outside the central nervous system Xsal-1 is expressed in the facio-acoustic ganglion and in the developing limb buds. The pattern of expression suggests that Xsal-1 might be under control of signals emanating from the notochord and/or the floor plate and that it might function in neuronal cell specification.

Zinc finger proteins in early Xenopus development.

The C2H2-type zinc finger motif defines a large super family of specific DNA and specific RNA binding proteins. Individual members of this protein family have been demonstrated to carry important regulatory functions in embryogenesis. We have isolated a large collection of C2H2-type zinc finger proteins from Xenopus laevis. Some of these proteins are highly conserved in evolution and found to be differentially expressed during embryonic development of the central nervous system. We also summarize our recent findings on the biochemical characterization of RNA and DNA binding activities in vitro for other Xenopus zinc finger proteins, which fall into structurally defined, distinct subfamilies.

Nucleoskeleton and nucleo-cytoplasmic transport in oocytes and early development of Xenopus laevis.

We use amphibian oocytes and eggs as favorite biological systems to study various cell biological phenomena. We have analyzed the role of the zinc finger protein TFIIIA and ribosomal protein L5 in nucleo-cytoplasmic transfer of 5S ribosomal RNA and report on the structural requirements of the 5S RNA for the interaction with TFIIIA. Furthermore, we have used the oocyte/egg system to analyze the kinetics of the posttranslational isoprenylation of oocyte nuclear lamin B3 and its fate during egg maturation. We demonstrate, that isoprenylation of newly synthesized lamins takes place in the oocyte cytoplasm before uptake into the nucleus and show, that the isoprene modifications alone are not sufficient to maintain stable association of lamins with nuclear envelope derived membranes in eggs. Finally, initial results of the identification of cis-acting sequence elements, involved in translational repression of lamin mRNAs in oocytes, are reported.

Accumulation of arsenic in yelloweye mullet (Aldrichetta forsteri) following oral administration of organoarsenic compounds and arsenate.

Groups of yelloweye mullet (Aldrichetta forsteri) were maintained for several weeks on diets containing one of a range of organoarsenic compounds (arsenobetaine, arsenocholine, 2-dimethylarsinylethanol, 2-dimethylarsinylacetic acid, 2-dimethylarsinothioylethanol) or arsenate. Fish fed 2-dimethylarsinylethanol, 2-dimethylarsinylacetic acid or 2-dimethylarsinothioylethanol showed no increase in arsenic concentrations in their muscle tissue, while fish fed arsenate showed small increases. The two groups of fish which received either arsenobetaine or arsenocholine had greatly elevated arsenic concentrations in their muscle tissue resulting from an estimated approximately 40% retention of ingested arsenic. Examination of the form of arsenic accumulated by fish fed arsenocholine showed that most of the arsenic (89%) was accumulated as arsenobetaine.

cDNA cloning of the developmentally regulated lamin LIII of Xenopus laevis.

Lamins are nucleoskeletal proteins which form intermediate type filaments in close association with the inner nuclear envelope membrane. Based on molecular and biochemical properties the lamins were grouped as type-A and type-B lamins, respectively. I have cloned the cDNA encoding lamin LIII of Xenopus which is the lamin protein present in oocyte nuclei and in cleavage nuclei. The data presented here indicate that a pool of maternal lamin LIII RNA is synthesized very early in oogenesis and that it continues to be present until gastrulation when the vast majority of the LIII RNA is degraded. Despite the similarities shared by all lamin proteins, the lamin LIII sequence neither possesses the features diagnostic for either type-A or type-B lamins nor does it show greater sequence similarity to one of the lamin types than to the other and thus it may represent a third type of lamin protein which may reflect its special function in oogenesis and early development.

The gene structure of Xenopus nuclear lamin A: a model for the evolution of A-type from B-type lamins by exon shuffling.

Nuclear lamins are intermediate filament (IF) type proteins that form a fibrillar network underlying the inner nuclear membrane. The existence of multiple subtypes of lamins in vertebrates has been interpreted in terms of functional specialization during cell division and differentiation. The structure of a gene encoding an A-type lamin of Xenopus laevis was analysed. Comparison with that of a B-type lamin of the same species shows remarkable conservation of the exon/intron pattern. In both genes the last exon, only 9-12 amino acids in length, encodes the complete information necessary for membrane targeting of lamins, i.e. a ras-related CaaX motif. The lamin A specific extension of the tail domain is encoded by a single additional exon. The 5' boundary of this exon coincides with the sequence divergence between human lamins A and C, for which an alternative splice mechanism had previously been suggested. Arguments are presented suggesting that B-type lamins represent the ancestral type of lamins and that A-type lamins derived there from by exon shuffling. The acquisition of the new exon might explain the different fates of A- and B-types lamins during cell division.