Search

We have utilized a rabbit reticulocyte lysate coupled transcription-translation system to express the large subunits of the clathrin associated protein-2 (AP-2) complex so that their individual functions may be studied separately. Appropriate folding of each subunit into N-terminal core and C-terminal appendage domains was confirmed by limited proteolysis. Translated beta 2 subunit bound to both assembled clathrin cages and immobilized clathrin trimers, confirming and extending earlier studies with preparations obtained by chemical denaturation-renaturation. Translated alpha a exhibited rapid, reversible and specific binding to clathrin cages. As with native AP-2, proteolysis of alpha a bound to clathrin cages released the appendages, while cores were retained. Further digestion revealed a approximately 29-kDa alpha a clathrin-binding fragment that remained tightly cage-associated. Translated alpha a also bound to immobilized clathrin trimers, although with greater sensitivity to increasing pH than the translated beta 2 subunit. Clathrin binding by both the alpha and beta subunits is consistent with a bivalent cross-linking model for lattice assembly (Keen, J. H. (1987) Cell Biol. 105, 1989). It also raises the possibility that the alpha-clathrin interaction may have other consequences, such as modulation of lattice stability or shape, or other alpha functions.

AP-3 (AP180) in rat sympathetic neurons maintained in culture was analyzed by pulse-chase labeling with [35S]methionine to look for post-translational modifications. At early times, two lower molecular weight precursors of the mature species were detected. By 10 min, all of the AP-3 was found in the mature form which is stable for at least 9 h. We show here that at least one of these processing events is due to the addition of O-linked N-acetylglucosamine (GlcNAc) which is present on the mature form of the protein. Wheat germ agglutinin, a GlcNAc-specific probe, bound to AP-3 and the binding was blocked by excess GlcNAc but not by excess mannose. Purified AP-3, and AP-3 in coated vesicles derived from bovine brain, served as substrates for beta-D-galactosyltransferase which is specific for terminal GlcNAc residues. Analysis of the disaccharide released by beta-elimination indicated that single GlcNAc residues are attached to AP-3 through an O-glycosidic linkage to threonine or serine residues. In vivo 32P-labeled AP-3, the result of serine phosphorylation (Keen, J. H., and Black, M.M. (1986) J. Cell Biol. 102, 1325-1333), bound to wheat germ agglutinin-Sepharose indicating that phosphorylation and glycosylation can occur simultaneously on the same molecule. Both modifications have been mapped to the central 50-kDa structural domain that is responsible for the anomalous migration of AP-3. Consistent with localization to the nonclathrin binding domain, the O-GlcNAc modification does not play a discernible role in the interaction of AP-3 with clathrin.

The Diabetes Control and Complications Trial (DCCT) provided much information towards settling the long-running controversy about the effectiveness of improving control of diabetes on the risk of its major complications. With the appearance or the advance of clinically significant retinopathy as its major outcome variable, DCCT randomised 1,441 insulin-dependent diabetic patients to conventional or intensified control groups. In both primary prevention and secondary intervention arms of the trial, intensified control reduced retinopathy risk by half or more, and also reduced nephropathy and neuropathy risks--however, risk of severe hypoglycaemic episodes was increased about three-fold. By contrast, there were no differences in quality of life, neurocognitive or emotional assessments between the two groups. The application of trial findings to 'real life' care is considered.

Clathrin is the structural protein of coated membranes involved in receptor-mediated endocytosis and aspects of Golgi sorting in eukaryotic cells. We have now detected a stoichiometric complex of clathrin with a novel protein of M(r) approximately 100,000 (100K) in lysates of different mammalian cells. Formation of the complex, which also includes the 70K heat-shock protein Hsc70, occurs within 15 min of synthesis. The 100K protein has been identified as valosin-containing protein (VCP; ref. 1), an early substrate for tyrosine phosphorylation on T-cell receptor activation. Further, VCP is the mammalian homologue of yeast Cdc48p (ref. 3) and is a member of a larger gene family that includes putative ATP-binding proteins involved in vesicle transport and fusion, 26S proteasome function, regulation of the expression of human immunodeficiency virus, and assembly of peroxisomes. The association with clathrin and the morphological and catalytic similarity to the chaperonin proteins indicate that VCP may modulate protein-protein interactions in membrane transport processes.

The controversy concerning certain deposits which may or may not belong to this interglacial is presented in Chapter 7. The account which follows adopts the orthodox view that there is sufficient evidence, notably of a biological nature, to assign most if not all of these deposits to the Ipswichian. While the majority of sites referred to this temperate stage on the basis of their fossil flora and fauna have been in East Anglia, a scattering has been found in other parts of the British Isles (Figure 8.1).

The type locality for this stage, which consisted of stadial and interstadial environments, has been designated as Four Ashes in southern Staffordshire (Mitchell et al. 1973a) (Figures 9.1 and 9.11). The stage name was derived from that of the Devenses, a tribe of ancient Britons which inhabited the region around Four Ashes (Shotton 1977b). The orthodox view is that the Devensian followed the Ipswichian Interglacial and began around 115000 BP (Table 1.1). It has been formally divided into Early (pre-50000 BP), Middle (50 000–26 000 BP) and Late (26 000–10 000 BP) periods (Mitchell et al. 1973a). However, as Shotton (1977b) has pointed out, the start of the Devensian has not been easy to delimit. This age is beyond the range of radiocarbon assay, although some U-series dates and amino-acid ratios have provided pointers to the commencement of Devensian time. Moreover, biostratigraphic evidence of the transition from the Ipswichian to the Devensian is scanty. Indeed, as Lowe and Walker (1984) have stated, if evidence in localities outside the British Isles is considered (notably from deep-sea and ice cores, loess stratigraphy, and pollen diagrams) (Chapter 1), a more complex sequence of environmental change is suggested, with the timing of the onset of the Devensian equivocal. It appears that a deterioration in climate occurred about 115 000 BP, probably equivalent to Oxygen Isotope Substage 5d. This was followed by a climatic amelioration of either interstadial or interglacial rank circa 100000 BP (Substage 5c), then a cooling episode around 90000 BP (Substage 5b). An interstadial or interglacial environment ensued once again about 80000 BP (Substage 5a). A significant cooling of climate is recorded for 80000–65 000 BP, which may equate with Stage 4 (Figure 1.2(a)). Thus, the Devensian could have begun about 115 000 BP, with the temperate episodes noted above being interstadials. Alternatively, these climatic ameliorations could have been post-Ipswichian and pre-Flandrian inter-glacials, and the last cold stage could have commenced with the marked cooling circa 80000 BP (section 9.1.11).

To compare the effects of placebo and GLA on the course of mild diabetic neuropathy over 1 yr.We entered 111 patients with mild diabetic neuropathy from seven centers into a randomized, double-blind, placebo-controlled parallel study of GLA at a dose of 480 mg/day. MNCV, SNAP, CMAP, hot and cold thresholds, sensation, tendon reflexes, and muscle strength were assessed by standard tests in upper and lower limbs.For all 16 parameters, the change over 1 yr in response to GLA was more favorable than the change with placebo, and for 13 parameters, the difference was statistically significant. Sex, age, and type of diabetes did not influence the result, but treatment was more effective in relatively well-controlled than in poorly-controlled diabetic patients.GLA had a beneficial effect on the course of diabetic neuropathy.

Appropriate nomenclature for the most recent Pleistocene stage has generated more discussion than that pertaining to the naming of any other division of this rank (Mangerud et al. 1974; Paepe et al. 1976; Hyvarinen 1978; Mangerud and Berglund 1978; West 1979). The present interglacial has been termed the Flandrian after a marine transgression within its timespan which has been identified on the Flemish coastal plain. With a lower limit of 10 000 radiocarbon years BP, the Flandrian usually has been considered equivalent to the formally designated Holocene Series and the informal Postglacial episode. The use of Flandrian has been recommended in the British Isles, although for Ireland the term Littletonian has been presented as synonymous (Mitchell et al. 1973a) (Table 10.1).

The duration of the Early Pleistocene in the British Isles has not been quantified. However, reference to palaeomagnetic and radiometric data from other areas suggests that it was in progress for at least 800000 years. In this account the stages referred to the Lower Pleistocene follow the scheme of Stuart (1982), who has justified the inclusion of the Pre-Pastonian and Pastonian in a Lower Pleistocene division (Table 1.1) on vertebrate faunal evidence. West (1980a) has noted that certain characteristics of Pastonian pollen assemblages link them to Early Pleistocene temperate-stage floras. However, in a scheme for East Anglia (West 1980b) the Pastonian and PrePastonian stages have been designated as Middle Pleistocene. Sites referred to within this chapter are shown in Figure 3.1.

Most of the evidence concerning the remaining Lower Pleistocene stages (sensu Stuart 1982) and those of the subsequent early part of the Middle Pleistocene (Table 1.1) has come from the Norfolk and Suffolk coasts. Of especial relevance has been the north and north-east Norfolk coast between Weybourne and Happisburgh, while the north-east coast of Suffolk between Corton and Southwold has also yielded important information. In general, the older deposits in this sequence have been located either beneath or at the level of the current foreshore, with the younger ones occurring towards the base of the cliffs; the latter being composed mainly of Anglian glacial material (Chapter 5). Deposition of these late Lower Pleistocene and early Middle Pleistocene sediments included the final infili of the Crag Basin, which may have taken place in part behind a Wadden barrier-island complex (West 1980a).

The scheme of Pleistocene stages in the British Isles of Mitchell et al. (1973a) (Table 1.1), assigned time between the Hoxnian and Ipswichian interglacials to the Wolstonian Cold Stage. It is noted in Chapter 5 that a dispute exists regarding the status of the Wolstonian Glaciation; deposits conventionally referred to it being considered of Anglian age by some investigators. It is also stated in Chapter 6 that evidence is now available for an interglacial stage between those of the Hoxnian and Ipswichian. Against this backdrop, the account which follows firstly presents details of the orthodox Wolstonian sequence, and secondly, examines data which point to the existence of at least one substantial additional temperate episode during the timespan under consideration. Sites relevant to these issues are shown in Figures 7.1 and 7.4.

Geomorphological, stratigraphical, pedological and palaeontological evidence has demonstrated that following the temperate environments which pertained during what may have been the final interglacial of the Cromerian Complex, cold conditions of the Anglian Stage affected the British Isles. As will be revealed below, most information concerning this Middle Pleistocene stage has accrued from East Anglia, with successions elsewhere traditionally considered to contain little or no representation of it. In recent years, however, the latter view has changed. An important consequence of this has been a questioning of the status of the Wolstonian Glaciation, formally designated as later in time (Mitchell et al. 1973a) (Table 1.1). Localities with deposits and/or landforms referred to the Anglian are shown in Figure 5.1.

Miocene and Pliocene deposits are not extensive in the British Isles. However, Walsh et al. (1972) have demonstrated that most areas have minor occurrences of sediments and evidence of weathering related to this timespan (Figure 2.1). As a result, knowledge of environments during the approximately 21 million years of these epochs is limited. The paucity of Neogene (Table 1.1) rocks is largely attributable to tectonic activity associated with the convergence of the African and European plates. Miocene deformation was not uniform. Scotland, northern England, Wales and parts of Ireland were uplifted, intervening areas downwarped; a tectonic pattern that remained operative in the Pliocene. Erosion was dominant, with the sea seemingly playing a major role in landscape evolution (Owen 1976). Miocene and Pliocene environments were quite different from those of the Pleistocene. This difference was associated with a significant climatic change at the end of the Pliocene, information concerning which is essential for an understanding of subsequent events. Localities referred to within this chapter are shown in Figure 2.1.

This interglacial has been defined by West (1956) following an investigation of the palaeobotany of lacustrine deposits overlying chalky (Lowestoft) till at Hoxne in Suffolk. Apart from the type site at Hoxne, a number of other localities of this age have been described in East Anglia, while analogous deposits have been recorded from the Thames Valley and its environs, the English Midlands, Sussex, Lincolnshire, Wales, Scotland and Ireland (Figure 6.1).

AP-2 and AP-3 are cellular proteins that drive the in vitro polymerization of clathrin triskelia into cage structures. The interaction of these two types of assembly proteins (APs) with preassembled clathrin cages has been studied in order to identify the sites on the triskelia required for binding. Comparing binding of the APs to intact or to proteolytically clipped cages, we attempted to distinguish between binding to the terminal domain, the globular end of the heavy chain, and binding to the hub of the clathrin triskelia, the portion that remains assembled after trypsin treatment. AP-3 binds to intact clathrin cages but not to those that were treated with trypsin. AP-3 also bound to cages consisting solely of clathrin heavy chains; proteolysis of these cages also eliminated AP-3 binding. In addition, AP-3 did not bind to either isolated hubs or terminal domains that had been immobilized on Sepharose. These data indicate that clathrin light chains are not required for binding of AP-3, and that neither terminal domain nor hubs alone will suffice. However, an intact heavy chain is both necessary and sufficient for the binding of AP-3. Previous work has demonstrated one binding site for AP-2 on proteolyzed cages containing only clathrin hubs; the existence of a second binding site associated with the terminal domain was hypothesized. Here we provide direct evidence for recognition by AP-2 of isolated terminal domains immobilized on Sepharose and show that the core of the AP-2 molecule is responsible for this interaction. These results provide the first demonstration of a functional role for the conserved terminal domain of the clathrin heavy chain.

Diagnostic criteria for diabetes mellitus (DM) and impaired glucose tolerance from oral glucose tolerance test results in adults are reviewed in the epidemiological context, highlighting the residual differences between World Health Organization (WHO) and National Diabetes Data Group (NDDG) glycemie criteria with respect to the diagnosis of gestational diabetes. Although the value of the diagnosis of DM (WHO/NDDG criteria) in pregnancy is not called into question, attention is drawn to the paucity of evidence linking lesser degrees of glucose intolerance with significant disturbance of pregnancy outcome when confounding variables such as maternal age, adiposity, and parity are allowed for. It is in the area of the detection and treatment of these lesser degrees of glucose intolerance in pregnancy that serious questions of the detriment-to-benefit ratio arise. A population-based multiethnic multicultural inquiry into diagnostic methodology and criteria in pregnancy is proposed, jointly sponsored by the WHO and the International Diabetes Federation, extending, if possible, to a controlled clinical trial of the effects of intervention.

We have studied the organization of clathrin during its transport in axons. Using immunoprecipitation techniques we have confirmed earlier findings that clathrin is transported as part of slow component b, but we also detect small amounts of clathrin in fast component. As fast component is known to correspond to the transport of membraneous material, including coated vesicle membrane components, our findings suggest that some clathrin in axons undergoes transport in the form of coated membranes and that a portion of the clathrin delivered to axons and axon terminals arrives by way of fast component. The organizational form of clathrin in slow component b (SCb) was examined in more detail, as it is thought to represent a non-membrane-associated species, is relatively long-lived, and at any instant represents the major transport species in axons. We used nondenaturing immunoprecipitation methods with stringent wash procedures to identify other SCb proteins that interact with clathrin. The immunoprecipitates contained major labeled bands that corresponded to clathrin heavy and light chains, along with a prominent 70-kDa band and several minor bands that ranged in apparent Mr from 70,000 to 150,000; the 70-kDa band was shown to be the ATP-dependent uncoating protein by two-dimensional gel electrophoresis. A very similar profile of polypeptides was also immunoprecipitated from extracts of cultured neurons. The results from a variety of control immunoprecipitations, including the use of antisera preadsorbed with purified clathrin trimers or clathrin light chains, indicate that coprecipitation of clathrin and uncoating protein with the other 70,000–150,000-Da polypeptides from SCb reflects specific interactions. Including exogenous uncoating protein in the lysis buffer had no detectable effect on the levels of endogenous uncoating protein recovered in the immunoprecipitates, indicating that complexes of clathrin, uncoating protein, and the other coimmunoprecipitating SCb protein existed in the intact neurons prior to lysis. Finally, a specific and functional association is further supported by the release of uncoating protein, but not the other 70,000– 150,000-Da polypeptides, from the immunoprecipitated complexes on the addition of ATP. Collectively, these observations provide the first direct evidence of interaction between clathrin and uncoating protein in intact cells, lend strong support to the concept that uncoating protein plays an intimate role in clathrin dynamics within cells, and reveal a family of 70,000–150,000-Da polypeptides that form a stable nonmembranous association with clathrin in intact cells.

The domains on clathrin responsible for interaction with the plasma membrane-associated assembly protein AP-2 have been studied using a novel cage binding assay. AP-2 bound to pure clathrin cages but not to coat structures already containing AP that had been prepared by coassembly. Binding to preassembled cages also occurred in the presence of elevated Tris-HCl concentrations (greater than or equal to 200 mM) which block AP-2 interactions with free clathrin. AP-2 interactions with assembled cages could also be distinguished from AP-2 binding to clathrin trimers by sodium tripolyphosphate (NaPPPi), which binds to the alpha subunit of AP-2 (Beck, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4442-4447). At concentrations of 1-5 mM, NaPPPi blocked clathrin-triskelion binding; in contrast, interactions with cages persisted in the presence of 25 mM NaPPPi. To begin to identify the region(s) of the clathrin molecule important in recognition by AP-2, clathrin cages were proteolyzed to remove heavy chain terminal domains and portions of the distal leg as well as all of the light chains. AP-2 bound to these "clipped cages"; however, unlike the interaction with native cages, binding of AP-2 to clipped cages was sensitive to the lower concentrations of both Tris-HCl and NaPPPi which disrupt interactions of AP-2 with clathrin trimers. Reconstitution of the clipped cages with clathrin light chains did not restore resistance of AP-2 binding to Tris-HCl. We conclude that one binding site for AP-2 resides on the hub and/or proximal part of the clathrin triskelion whereas a second site is likely to involve the terminal domain and/or distal leg; the second site is manifested only in the assembled lattice structure. We suggest that these two distinct binding interactions may be mediated by the two unique large subunits within the AP-2 complex, acting sequentially during assembly.

A self-association reaction involving the plasma membrane-associated clathrin assembly protein AP-2 has been detected by incubating AP-2 alone under solution conditions that would favor the assembly of complete coat structures if clathrin were present. Self-association was rapid, unaffected by nonionic detergents, readily reversible, and gave rise to sedimentable aggregates. Only the AP subtype AP-2 exhibited self-association: the structurally or functionally related assembly proteins AP-1 and AP-3 and unrelated proteins neither self-associated nor were incorporated into the AP-2 aggregate. AP-2 interactions responsible for self-association were of high affinity, with an apparent Kd of approximately 10(-8)M. By proteolytic dissection, the self-association domain was localized to the core of the molecule containing the intact 50- and 16-kDa polypeptides in association with the truncated 60-66-kDa moieties of the parent alpha/beta polypeptides. Self-association of the intact AP-2 molecule was pH-dependent, exhibiting an apparent pKa approximately 7.4. While it is unlikely that the large AP-2 aggregates formed in solution are themselves biologically relevant structures, the AP-2 interactions involved in their formation have properties consistent with their occurrence in intact cells and thus may be important in cellular functions of the plasma membrane-localized assembly protein.

Three independently isolated clathrin-associated proteins have been reported that have molecular weights of approximately 155,000-185,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis: the 155K protein (Keen, J. H., and Black, M. M. (1986) J. Cell Biol. 102, 1325-1333), AP 180 (Ahle, S., and Ungewickell, E. (1986) EMBO J. 5, 3143-3149), and NP185 (Kohtz, D. S., and Puszkin, S. (1988) J. Biol. Chem. 263, 7418-7425). Using two-dimensional isoelectric focusing polyacrylamide gel electrophoresis and one- and two-dimensional immunoblots with two different monoclonal antibodies, we show that these three proteins are identical. The term AP-3 is used to denote this protein. A preliminary analysis of the domain structure of AP-3 was done by controlled proteolysis. Trypsin treatment of AP-3 yields two distinct classes of products. The larger fragments obtained (100,000-135,000 apparent Mr) are acidic and behave anomalously on gel electrophoresis, yielding aberrantly high Mr and exhibiting poor dye binding; these characteristics are shared with intact AP-3. Trypsin also generates a smaller neutral species of approximately 30,000 Da which migrates appropriately on sodium dodecyl sulfate-gel electrophoresis, binds dye comparatively strongly, and behaves as a monomeric globular species in solution. In addition, this species, which is also released by a variety of other proteases, binds specifically and reversibly to clathrin-Sepharose, identifying it as a clathrin recognition domain.

Several components of the phosphoinositide cycle have been found to interact specifically and at physiological concentrations with the plasma membrane-associated clathrin assembly (adaptor) protein AP-2. These include phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate, which are present at the plasma membrane, as well as other polyphosphoinositols. ATP and other polyphosphate molecules complete with the polyphosphoinositols, however, they are at least 80-fold less potent. Also, the effect of ATP, unlike the polyphosphoinositols, is blocked by physiological concentrations of Mg2+. Photoaffinity labeling of AP-2 by [alpha-32P]8-azidoadenosine 5'-triphosphate and its competition by polyphosphoinositols has been used to identify the alpha subunit of the AP-2 complex as the site of specific interaction with the polyphosphoinositols and to confirm direct ultrafiltration binding experiments. Proteolytic dissection of the labeled AP-2 demonstrated that binding occurred exclusively on the N-terminal portion of the alpha subunit. Interaction of purified AP-2 with sub-microM concentrations of polyphosphoinositols has inhibitory effects on a novel AP-2 self-association described in the accompanying paper (Beck, K. A., and Keen, J. H., J. Biol. Chem. 266, 4437-4441), and at higher concentrations on the binding of AP-2 to dissociated clathrin trimers as well as AP-2-mediated clathrin coat assembly. Review of the literature shows that several physiological stimuli that are known to result in increased coat pit formation in intact cells correlate with increased phosphoinositide turnover. These in vivo correlations and the in vitro observations reported here suggest that coated membrane and phosphoinositide cycles may be interdependent within cells.

The 497 members of the London Cohort of the WHO Multinational Study of Vascular Disease in Diabetics have been followed for mortality from 1975 to 1987. During this period 92 patients died. The most common cause of death was myocardial infarction: 36 (39.1%) deaths, heart disease was responsible for 51.1% of deaths and all cardiovascular disease for 55.4%. Neoplastic disease accounted for 25% of the deaths and diabetic nephropathy for 5.4%. Age-standardised mortality rates were higher in men than in women in both Type 1 (insulin-dependent) diabetes and Type 2 (non-insulin-dependent) diabetes. Standardised mortality ratios for the first and second five year follow-up periods were higher for men than for women in Type 2 diabetes but were higher for women than men in Type 1. The results suggest that the female survival advantage seen in the general population may persist in Type 2 but not in Type 1 diabetes.

PERSPECTIVES AND SUMMARY. . ....... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415 CLATHRIN . . . . . ...... . . . . . . . . .. . . . . .. .... .. . . . . . . . . ....... . .. . . . . . . . . . . . . . . . . . . . . . ...... . . . . 4 1 7 Clathrin Heavy Chains 417 Clathrin Light Chains 417 Clathrin Trimer/Triskelion...... ......... ...... ........ . . . . . . . . ....... . . . . . . . . . 419 APs: ASSEMBLY PROTEINS/ADAPTOR PROTEINS/ASSOCIATED PROTEINS.... 423 AP-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423 AP-I and AP1801AP-3 . 426 AP Activities 427 AP-Membrane Interactions 429 UNCOATING ATPase/Hsc70. . . . ...... . . . . . 431 Uncoating ATPase is Hsc70 431 Functions of the Hsc70lUncoating ATPase 433

Clathrin-coated vesicles, involved in endocytosis and Golgi processing, have a surface lattice containing clathrin triskelia and stoichiometric amounts of additional components termed {open_quotes}assembly proteins,{close_quotes} or APs. The AP form at the plasma membrane, AP2, is composed of two large subunits of 100-115 kDa, denoted AP2{alpha} and AP2{beta}, a medium chain of 50 kDa, designated AP50, and a small chain. We have determined human chromosomal locations of genes for a large AP2{beta} (CLAPB1) and a medium (CLAPM1) AP subunit and of a novel clathrin-binding protein, VCP, that binds clathrin simultaneously with A1`s. Chromosomal in situ hybridization of a human genomic clone demonstrated that the CLAPM1 gene mapped to chromosome region 3q28. The gene for the CLAPB1 large subunit was mapped to 17q11.2-q12 by PCR amplification of an AP2{beta} fragment from a panel of rodent-human hybrid DNAs. To map the human VCP sequence, a human-specific probe was made by RT-PCR of human mRNA using oligonucleotide primers from conserved regions of the porcine sequence. The amplified human fragment served as probe on Southern blots of hybrid DNAs to determine that the human VCP locus maps to chromosome region 9pter-q34. 13 refs., 2 figs.