The insulin-like growth factors are single-chain polypeptides which promote cell multiplication in vitro. Their role in mammalian development is uncertain, although they have been implicated as modulators of cell growth and differentiation. We present evidence that the human IGF-II gene has at least two promoters, and their expression may be developmentally controlled in the liver. Most of the IGF-II transcripts in the fetal organs examined are derived from a promoter which is different to that used for most adult liver IGF-II mRNAs. Steady-state levels of IGF-II transcripts are seen to be dramatically reduced in organs of adult rather than fetal origin. This observation is apparently not linked to promoter usage and therefore suggests a second level of transcriptional control.

In addition, we show that an alternative splicing event at an intron/exon boundary, which results in an mRNA with an altered coding potential, is not developmentally regulated. This variant IGF-II mRNA is coexpressed with the major species of IGF-II at a low, but constant, ratio in all fetal and adult organs examined.

The insulin-like growth factors (IGFs) are small proteins that promote cell multiplication. They are found in fetal and adult serum bound to carrier proteins, and these circulating IGFs may have general endocrine functions. However, there is evidence that IGFs may also control local tissue growth and differentiation during embryonic and postnatal mammalian growth in an autocrine or paracrine fashion (for reviews see Sara & Hall, 1984 and Gluckman, 1986). There are two major forms of IGFs, IGF-I and IGF-II, 70 and 67 amino acids in size, respectively, which are closely homologous. Their primary structure also shows similarities to insulin.

Human IGF-II cDNA probes have been used to examine the expression of IGF-II mRNA in organs of the late first trimester human embryo (Scott et al. 1985; Hyldahl, Engstrom & Schofield, 1986). Although the overall pattern of transcripts is similar in different organs, the relative amounts of different size classes of transcript seem to vary between very low in fetal brain to very high in the liver. IGF-II transcripts are less abundant in adult organs and, uniquely, IGF-II mRNA from adult liver shows a single major transcript (Scott et al. 1985; Irminger, Rosen, Humbel & Villa-Komaroff, 1987).

The rat and human IGF-II genes have been isolated. They are both single-copy genes and have the same exon size and domain structure in the coding region, which lies 20kb 3’ of the insulin gene. The consensus structure of the human gene is shown diagrammatically in Fig. 1 (Tricoli et al. 1984; Dull, Gray, Hayflick & Ullrich, 1984; Bell et al. 1985; de Pagter-Holthuizen et al. 1986, 1987; Soares et al. 1986; Frunzio et al. 1986). Transcripts containing the 5’non-coding exons E-l and E-2 have only been isolated from human adult liver cDNA libraries and equivalent regions have not been identified in the rat. Two promoters are active upstream of exons El(l) and El(2) in rat tissues and the relative use of these two promoters does not appear to change with developmental stage (Soares et al. 1986; Frunzio et al. 1986). Human transcripts from these promoters have not previously been identified, although a homologous

Fig. 1.

Genomic organization of human IGF-II gene. (A) The location of exons, Alu repeat and the insulin gene is compiled from Dull et al. 1984; Bell et al. 1985; de Pagter-Holthuizen et al. 1986, 1987; and our results. Exon El (2) is located by analogy with the rat. The 3’end of E4 is unknown. A-C are three published cDNA clones. A and B are found in adult liver, differing by a 9 bp addition at the start of E3 (shown as a black sector in Fig. 1; Jansen et al. 1985). C has been isolated from a human hepatoma cDNA library (de Pagter-Holthuizen et al. 1987). D represents a novel cDNA clone whose isolation is reported in this paper. (B) The location of three bacteriophage genomic clones which we have isolated. EcoRI (E) and SalI (S) restriction enzyme sites are marked.

Fig. 1.

Genomic organization of human IGF-II gene. (A) The location of exons, Alu repeat and the insulin gene is compiled from Dull et al. 1984; Bell et al. 1985; de Pagter-Holthuizen et al. 1986, 1987; and our results. Exon El (2) is located by analogy with the rat. The 3’end of E4 is unknown. A-C are three published cDNA clones. A and B are found in adult liver, differing by a 9 bp addition at the start of E3 (shown as a black sector in Fig. 1; Jansen et al. 1985). C has been isolated from a human hepatoma cDNA library (de Pagter-Holthuizen et al. 1987). D represents a novel cDNA clone whose isolation is reported in this paper. (B) The location of three bacteriophage genomic clones which we have isolated. EcoRI (E) and SalI (S) restriction enzyme sites are marked.

DNA region has been identified in genomic clones (Dull et al. 1984).

We have used SI nuclease protection analysis of mRNA with newly isolated genomic and cDNA clones to show for the first time that the majority of fetal IGF-II transcripts initiate from a promoter at exon El(l). Further, it is shown that this promoter is different to that used in adult liver. This change of promoter usage can be interpreted as a developmental switch in IGF-II expression in the liver. Also, we demonstrate that an alternative splicing event, capable of generating a variant form of IGF-II, occurs in both fetal and adult transcripts.

Genomic libraries

A human bacteriophage EMBL3 genomic library made from DNA isolated from the blood of a Factor IX-deficient patient was kindly supplied by Dr Anne Bentley, Sir William Dunn School of Pathology, Oxford, UK. This was screened with a 0-8 kb Pstl fragment derived from a human adult liver IGF-II cDNA clone phigf2 (Bell et al. 1984). A 1-6kb BamHI fragment from a subclone of the human insulin gene phins2l4 was used to locate that gene. phigf2 and phins2lA were kindly supplied by Dr James Scott, Clinical Research Centre, Harrow, UK. Positive clones were purified to homogeneity and restriction enzyme mapped by standard techniques.

cDNA clones

A Hep G2 (human hepatoma cell line; Aden et al. 1979) cDNA library cloned with synthetic EcoRI linkers into lambda gtlO was kindly supplied by Dr James Scott, Clinical Research Centre, Harrow, UK. This was probed with the 0-8kb Pstl fragment described above. Positive clones were purified to homogeneity, restriction enzyme mapped and then subcloned into pUC8 for chemical DNA sequencing (Maxam & Gilbert, 1977), or into M13mp8 for sequencing by the chain termination method (Sanger. Nicklin & Coulsen, 1977).

RNA extraction

Total RNA was extracted from 8- to 12-week fetal tissue (collected and staged as in Shi et al. 1985), frozen liver from a 10-year-old adult male, or cultured cells by the guanidine thiocyanate method (method of Chirgwin. Przybyla. McDonald & Rutter, 1979 as modified in Hyldahl et al. 1986).

SI analysis

SI analysis was carried out by a modification of the method of Favaloro, Treisman & Kamen (1980). 5-−0μg total RNA was hybridized to excess 5’end-labelled double-stranded DNA probes (approximately 100 000 cts min-1) in a 10 μl reaction containing 80% (v/v) formamide, 40mM-Pipes pH 6·4, 0·4M-NaCl, 1 mM-EDTA under paraffin oil at 52°C overnight, following heating to 80°C for 10 min to denature the probe. Subsequently, the reaction tube was placed on ice and 300μl of ice-cold SI buffer (30mM-sodium acetate pH 4·6, 4mM-zinc sulphate, 0-28M-NaCl, 5 % (v/v) glycerol containing 15 μg sonicated denatured salmon sperm DNA as carrier. 60 units SI nuclease) was added. Tubes were incubated at 30°C for 1 h and the reaction terminated by the addition of 100 μl 15 mM-EDTA pH 7·9 containing 30 pg tRNA carrier, followed by ethanol precipitation. Reaction products were separated on 7 M-urea-containing polyacrylamide gels. Deviations from this protocol are noted in figure legends or text.

Primer extension

5pg fetal or adult liver mRNA was incubated with Ing 5’ end-labelled oligonucleotide (5’AGAGCGCGGGCAGG-CGTGGG 3’) in 10μl of SI hybridization buffer (minus formamide) at 90°C for 4min, followed by 48°C for 60min, and then cooling on ice. The solution was adjusted to 50mM-Tris-HCl pH 8·3 (at 42°C), 10mM-DTT, 6mM-MgCh, 0·5 mM each deoxyribonucleotide triphosphates plus 5 units reverse transcriptase (Life Sciences; Northumbria Biologicals Ltd, Northumberland, UK) and incubated at 42°C for 60min. After ethanol precipitation, products were analysed as for SI analysis.

IGF-II transcripts have alternative 5’ noncoding sequences

Northern blot analysis of fetal and adult mRNAs indicated that most fetal organs contained several different-sized IGF-II transcripts and that adult liver contained a single, unique transcript (Scott et al. 1985; Irminger et al. 1987). To discover if these transcripts were generated by alternate splicing, an SI analysis of fetal, adult and neoplastic mRNA was performed (Fig. 2). The probe was prepared from a subclone of an adult liver IGF-II cDNA (phigf2, Bell et al. 1984). Adult liver RNA protected a fragment of 513 bases (band C), which was equivalent to the entire probe less the vector polylinker region. In contrast, all the other mRNAs of fetal and neoplastic origin showed a major protected band of 282 bases (band B). This observation suggests that the majority of IGF-II mRNAs, in most of the samples tested, differed from adult liver mRNA in the 5’ noncoding sequence spliced onto exon 2. However, a minority of mRNAs from fetal liver, spinal cord, adrenal and kidney clearly protected the adult probe throughout its 5’ noncoding region.

Fig. 2.

SI nuclease analysis using an adult liver cDNA probe. SI analysis of mRNA from adult liver (10-year-old male), human neoplastic cell lines Hep G2 (hepatoma), BeWo (trophoblastic choriocarcinoma; Patillo & Gey, 1968), Gos4 (Wilms), Tera2EC (teratoma; Thompson et al. 1984) and Tera2diff (its retinoic acid-induced differentiated derivative) and nine fetal organs. Fetal organs were pooled except in the case of lung and spinal cord, which were from different individuals. The origin of the probe, labelled at a SalI site (see Fig. 4), and the protected fragments A, B and C are shown diagrammatically below. Size markers are end-labelled pBR322 fragments from a HinfI digest. The intensity of bands other than A, B and C could be diminished by changing hybridization or SI incubation temperatures, indicating them to be artefactual (result not shown). possibility of developmental regulation in liver transcription in the human.

Fig. 2.

SI nuclease analysis using an adult liver cDNA probe. SI analysis of mRNA from adult liver (10-year-old male), human neoplastic cell lines Hep G2 (hepatoma), BeWo (trophoblastic choriocarcinoma; Patillo & Gey, 1968), Gos4 (Wilms), Tera2EC (teratoma; Thompson et al. 1984) and Tera2diff (its retinoic acid-induced differentiated derivative) and nine fetal organs. Fetal organs were pooled except in the case of lung and spinal cord, which were from different individuals. The origin of the probe, labelled at a SalI site (see Fig. 4), and the protected fragments A, B and C are shown diagrammatically below. Size markers are end-labelled pBR322 fragments from a HinfI digest. The intensity of bands other than A, B and C could be diminished by changing hybridization or SI incubation temperatures, indicating them to be artefactual (result not shown). possibility of developmental regulation in liver transcription in the human.

In addition, a 127-base Sl-protected fragment was generated by all the mRNAs tested (Fig. 2, band A). This implies a discontinuity between some of the IGF-II mRNAs and the probe between exons 2 and 3. This most probably results from additional alternate splicing, as will be discussed later.

Exon El(l)-containing IGF-II transcripts and their abundance

In order to locate the novel 5’noncoding sequences that are spliced onto the coding exons in fetal mRNA, we have analysed new cDNA clones from a Hep G2 human hepatoma cell library. This library was chosen because both Northern blot and the SI analysis indicated that Hep G2 cells contained large quantities of IGF-II-specific mRNA with a splice discontinuity at the same position as fetal mRNAs. A convenient unique Hinfl site, located 11 nucleotides from the extreme 5’end of exon 2 and only 5 nucleotides from the initiating AUG codon, was used to divide the novel cDNA inserts into translated and nontranslated regions. The Hinfl fragments were hybridized to Southern blots of newly isolated, overlapping genomic clones, which contain both the genes for IGF-II and insulin and which cover around 46kbp of the genome. The 5’nontranslated fragment of each mapped to the 3’ extremity of the region shown as exon El(l) in Fig. 1, whilst downstream the sequences mapped to the previously reported exon positions E2, 3 and 4 (results not shown; see Dull et al. 1984; Bell et al. 1985; de Pagter-Holthuizen et al. 1986). Therefore, the origin of the 5’ nontranslated region in these clones is different from that of the adult liver cDNAs (Bell et al. 1984; Jansen et al. 1985), which map 6 kb upstream of the coding exons (exons E-l and E-2, Fig. 1).

To estimate the frequency with which the 5’ noncoding exon El(l) was represented in IGF-II transcripts, we have extended the SI analysis with a probe derived from a cDNA clone containing this novel region. Most of the IGF-II mRNA molecules of fetal, tumour or cell line origin contain this 5’ noncoding exon El(l) since they protect the entire probe fragment extending into noncoding regions, less vector sequences (Fig. 3, band B). Using adult liver mRNA, a smaller protected fragment of 143 bases is obtained (band A). This confirms the observation that IGF-II transcripts in adult liver contain alternate 5’ noncoding sequences (Fig. 2).

Fig. 3.

SI nuclease analysis using a Hep G2 derived cDNA. SI analysis of adult liver, fetal organ and neoplastic cell mRNA using an IGF-II cDNA clone containing exon El(l). The origin of the probe, labelled at an Avail site in exon E2, and protected fragments A, B and C is shown in the lower panel. All organs were fetal unless otherwise stated. SI nuclease digestion was carried out at 42°C rather than 30°C in an attempt to remove artefactual bands caused by the base composition of the probe. The species seen at 246 bp is artefactual since it is not detectable under more stringent conditions of SI nuclease digestion. Markers were an end-labelled set of 123 bp DNA fragments (Gibco BRL Ltd, Uxbridge, UK).

Fig. 3.

SI nuclease analysis using a Hep G2 derived cDNA. SI analysis of adult liver, fetal organ and neoplastic cell mRNA using an IGF-II cDNA clone containing exon El(l). The origin of the probe, labelled at an Avail site in exon E2, and protected fragments A, B and C is shown in the lower panel. All organs were fetal unless otherwise stated. SI nuclease digestion was carried out at 42°C rather than 30°C in an attempt to remove artefactual bands caused by the base composition of the probe. The species seen at 246 bp is artefactual since it is not detectable under more stringent conditions of SI nuclease digestion. Markers were an end-labelled set of 123 bp DNA fragments (Gibco BRL Ltd, Uxbridge, UK).

Location of promoter for mRNA species which contain El(l)

We have shown that the major fetal IGF-II mRNAs contain exon El(l) sequences which are contiguous with the coding region in exon E2. Since there is an active IGF-II promoter in the rat, which is situated immediately upstream of exon El(l), we were interested to know whether this promoter was used in human cells. This was addressed in two ways. A 20 base oligomer derived from near the 5’-end of the human sequence equivalent to exon El(l) was used in a primer extension experiment (Dull et al. 1984). A strong stop to extension occurred on fetal, but not adult; liver mRNA at about 95 bases (Fig. 4B). That this position could indeed be the start site for transcription of fetal liver IGF-II mRNAs was confirmed with an SI protection experiment using a genomic fragment 5’end-labelled within the exon El(l) and extending to an EcoRI site approximately 400 base pairs upstream. By comparison with a DNA sequencing ladder of the same fragment, it was shown that a 139- and 140-base doublet of Sl-protected bands could be seen with fetal, but not adult, liver mRNA (Fig. 4A), indicating that exon El(l) in the human is 1164/5 base pairs long, starting with either an A or G residue. This position corresponds almost exactly with the position of the rat El(l) cap site (Soares et al. 1986; Frunzio et al. 1986). Motifs typical of RNA polymerase II promoter sites are found around positions −25 (a TATAA box) and −88 (a CCATT box), indicating that human fetal IGF-II transcripts containing exon El(l) are initiated in this region (Fig. 5; McKnight & Tjian, 1986). Therefore, adult liver IGF-II transcripts, containing exons E-l and E-2, must be independently promoted from a region closer to the insulin gene, suggesting the

Fig. 4.

Location of the promoter for exon El(l). (A) SI analysis using a genomic fragment, represented diagrammatically beneath the figure, with fetal and adult liver RNA and yeast tRNA as a control. A DNA-sequencing ladder of the probe fragment (lanes cut off as overexposed) was used as size marker and to obtain the DNA sequence at the promoter. The TATAA region is boxed. Doublet at position A represents the A or G nucleotides at the start of exon El(l). (B) Primer extension product (PE) obtained with fetal liver RNA from a synthetic oligonucleotide (see diagram).

Fig. 4.

Location of the promoter for exon El(l). (A) SI analysis using a genomic fragment, represented diagrammatically beneath the figure, with fetal and adult liver RNA and yeast tRNA as a control. A DNA-sequencing ladder of the probe fragment (lanes cut off as overexposed) was used as size marker and to obtain the DNA sequence at the promoter. The TATAA region is boxed. Doublet at position A represents the A or G nucleotides at the start of exon El(l). (B) Primer extension product (PE) obtained with fetal liver RNA from a synthetic oligonucleotide (see diagram).

Fig. 5.

Genomic DNA sequence around the promoter for exon El(l). CCATT and TATAA elements are underlined. The SalI site was end-labelled for both DNA sequencing and SI analysis. The alternate cap nucleotides are starred above, the G residue being designated as +1. The cap site in the rat has been identified as either residue +5 (Soares et al. 1986) or +8 (Frunzio et al. 1986).

Fig. 5.

Genomic DNA sequence around the promoter for exon El(l). CCATT and TATAA elements are underlined. The SalI site was end-labelled for both DNA sequencing and SI analysis. The alternate cap nucleotides are starred above, the G residue being designated as +1. The cap site in the rat has been identified as either residue +5 (Soares et al. 1986) or +8 (Frunzio et al. 1986).

Variant IGF-II mRNA transcripts

Three out of four cDNA clones isolated from the Hep G2 library had an identical insertion of nine base pairs at the same position as that found in a previously reported IGF-II cDNA clone (Jansen et al. 1985; black segment in E3, Fig. 1). Such mRNA would potentially code for a variant form of IGF-II. Therefore, it was of interest to determine which organs express such a transcript and in what quantities. This was achieved by SI analysis using a probe prepared from a cDNA clone containing the extra sequence, 5’end-labelled at the Sall site in exon 3 (Fig. 6). Three protected fragments are apparent, the strongest (band A), shown as a doublet around 127 bases, representing the most abundant mRNA species which do not contain the extra nine bp. However, the 359-base protected fragment (band C), corresponding to all those mRNAs which are entirely colinear with the cDNA probe, clearly shows that there are IGF-II mRNAs with both the 5’noncoding exon El(l) and the additional nine base pairs. These mRNAs make up a substantial fraction of the total IGF-II mRNA in both fetal and adult organs. A third protected fragment of 287 bases is apparent (band B), especially in the solid Wilms’ tumour examined (Bema). This represents variant IGF-II transcripts whose 5’noncoding sequence must differ from that of the Hep G2 clone. The most likely explanation is that they are transcribed from the adult liver promoter although usage of a promoter at El(2) cannot be excluded.

Fig. 6.

SI analysis to establish abundance of variant IGF-II transcripts. The origin of the probe, a Hep G2-derived variant cDNA labelled at the Sall site in exon E3 and recut in plasmid sequences, together with that of the protected fragments A, B and C, is shown in the lower panel. 20μg of adult liver RNA was used; 5μg RNA in all other cases. All organ RNA samples were fetal unless specified and were taken from single separate individuals, with the exception of yolk sac. Markers as in Fig. 3.

Fig. 6.

SI analysis to establish abundance of variant IGF-II transcripts. The origin of the probe, a Hep G2-derived variant cDNA labelled at the Sall site in exon E3 and recut in plasmid sequences, together with that of the protected fragments A, B and C, is shown in the lower panel. 20μg of adult liver RNA was used; 5μg RNA in all other cases. All organ RNA samples were fetal unless specified and were taken from single separate individuals, with the exception of yolk sac. Markers as in Fig. 3.

In summary, there appears to be developmental regulation of the human IGF-II gene promoter usage in the liver. There is no evidence that this switch occurs in any other organ. However, levels of IGF-II transcripts are downregulated in all the organs of the adult, so this must be mediated by a separate controlling event. mRNAs coding for the insertion variant form of IGF-II are found in all organs and cell types that express the gene, there being no apparent tissue- or development-specific event controlling their transcription.

IGF-II transcripts differ at their 5’ ends

By cDNA cloning and SI analysis, we have shown for the first time that the majority of IGF-II transcripts in fetal organs and neoplastic cells comprises exon E1 (1) spliced onto the coding exon E2 (Fig. 1). In Contrast, adult liver mRNA transcripts contain alternate 5’ noncoding sequences (exons E−1, E−2 and E−3) and hence must initiate from a different promoter.

A recent report describes the isolation of cDNA clones from adult human thalamus (Irminger et al. 1987). Like the Hep G2 clones, 5’ non-coding sequences are derived from exon El(l). Hybridization with exon El(l)-specific probes showed that specific transcripts were present in adult thalamus, kidney and adrenal gland and in placenta. Therefore, exon El(l)-containing transcripts are definitely not confined to the fetus, occurring in other adult organs with the exception of liver.

Developmental regulation of IGF-II promoters

Recently it has been demonstrated that IGF-II transcription in the rat initiates from one or other of two promoters, preceding exons El(l) or El(2) (Fig. 1, see Soares et al. 1986; Frunzio et al. 1986). There is no evidence for developmental regulation in their use. Results of hybridization experiments using exonspecific probes indicate that IGF-II mRNA in both fetal and adult rat organs initiates predominantly from the El(2) promoter. The most 5’ of these promoters, El(l), is equivalent to that described in this paper as being used predominantly in human fetal organs, whereas we have no evidence so far that a promoter equivalent to El(2) is used in the human. The 5’ noncoding sequences found in adult human liver cDNA clones map to a position approximately 6 kb further 5’ of exon El(l) (Bell et al. 1985) and our SI analysis confirms that mRNA containing these sequences comprise the major species transcribed in this organ. Since we provide evidence that a promoter downstream of these sequences is used preferentially in fetal organs, there must be an alternative promoter used in adult liver, mapping much closer to the insulin gene. Recent data suggest that this is the case (de Pagter-Holthuizen et al. 1987). A rat equivalent of this promoter has not yet been found although its existence cannot be formally excluded.

It must therefore be concluded that regulation of expression of the IGF-II gene is different in human and rat organs since homologous promoters, El(l) and El(2), are being used differently by the two species. There is no evidence in the rat of the developmental switch in liver-specific promoter usage seen in humans which may be linked with the observation that there is no apparent homologue to the human adult liver-specific promoter. We do not have evidence for developmental regulation of IGF-II expression in any organ other than the liver. Indeed, Northern blot analysis indicates that probes from exons E−1 and E−2 hybridize only to the 5·3 kb transcript unique to adult liver, although an extensive analysis of other adult tissues has yet to be carried out (Scott et al. 1985; Irminger et al. 1987). Other genes have been described as having alternate promoters which are regulated in either a tissue-specific or developmentally specific manner (reviewed in Leff, Rosenfeld & Evans, 1986). It will be of interest to determine the basis of this control, for example, whether it is regulated by cis- or trans-acting elements.

Our results can be interpreted as indicating that there is a developmental switch in promoter usage in the liver from the fetus to the adult. Since we do not have any in situ hybridization data, we do not know whether this change occurs in a particular cell type or whether it represents a change in the cell populations which make up the liver. The consequence of such regulation is the expression of mRNAs which differ at the 5’end. Computer searches have failed to find any open reading frames within these sequences. Therefore, the functional significance of this change is unclear. Comparison of El(l) sequences between the human and rat reveal a homology which is much greater than would be expected in a noncoding region, implying a specific function. Whether this concerns mRNA stability or involves interactions with regulatory factors remains to be determined.

Variant IGF-II mRNA expression

A previous report (Jansen et al. 1985) described the isolation of an adult liver IGF-II cDNA clone with an insertion of nine base pairs in the coding region. This mRNA codes for a variant form of IGF-II which has been purified as a minor component from pooled human serum (R. E. Humbel, personal communication). A comparison of the cDNA sequence with that of a genomic clone suggests that variant mRNAs are generated by alternative splicing to an acceptor site, nine base pairs 5’ to that normally used at exon E3. However, the sequence in the intron at this position is GG, which therefore deviates from the canonical AG normally found at a 3’splice acceptor site, and which is identical to an apparently nonfunctional acceptor (Atweh et al. 1985). Since IGF-II is a single-copy gene (Jansen et al. 1985; unpublished observations), this raises the possibility that this alternative form of IGF-II is an allelic variant. We have isolated further cDNA clones with this additional sequence from the Hep G2 library and used them in SI analyses to show that mRNA coding for the variant IGF-II is not restricted to adult liver, being found at a low level in all organs and cell lines examined so far. Therefore, there is no evidence for either tissue or developmental-specific transcription of variant mRNA. Since at least twelve of these analyses were from discrete individuals and there is an absence of evidence for heterozygous advantage at this site, it is highly unlikely that the mRNA arises from an allelic variant. The genomic sequence of this region has been obtained from two alleles from separate individuals and, therefore, it is unlikely that a mutant splice acceptor site was cloned. The ability to splice to an altered sequence may be dependent on other, more distant, regions which are, as yet, unidentified.

IGF-ll transcript abundance versus protein levels

The decrease in steady-state levels of IGF-II transcripts in human adult liver relative to the fetus does not seem to reflect serum IGF-II levels, as these actually increase in the adult (Ashton, Zapf, Einschenk & MacKenzie, 1985). Such a discrepancy could be explained by postulating that the peptide is stabilized in adult serum, perhaps by association with a binding protein. Alternatively, all the IGF-II being produced in the adult liver may be secreted in an endocrine fashion into the bloodstream. The higher levels of IGF-II produced in the fetal liver may all be utilized locally by autocrine or paracrine mechanisms and never enter the circulation.

Serum levels of IGF-II in the rat fall precipitously after birth (Moses et al. 1980). There is, at present, no evidence for a rat equivalent of the human adult liver promoter. Therefore, if transcripts from the human adult liver promoter are responsible for the elevated serum levels of IGF-II, lack of such a promoter in the rat might explain why there is this difference in adult serum levels between species.

Levels of IGF-II transcripts in other adult organs in the rat fall considerably compared with the fetus and a limited analysis, by Northern blotting, indicates that this may be true in humans (Brown et al. 1986; Scott et al. 1985; Irminger et al. 1987). This can be considered as another example of developmental regulation, but since there is no evidence of a change in promoter usage, it is more likely to be due to either changes in tranj-acting factors affecting promoter strength or mRNA stability.

In conclusion, considerable evidence exists for developmental regulation of IGF-II in humans. In situ hybridization studies are under way to confirm and extend these observations. Studies with the cloned promoter in different cell types will be used to determine at what levels this regulation is operating. Apparent differences in rat and human IGF-II expression signal caution in making comparisons between the two systems.

Amanda Lee and Lynne Richardson are thanked for their excellent technical assistance, and Brian Hopkins and Chris Graham for help in organ RNA preparation. We wish to thank Dr James Scott (CRC, Harrow, UK) for the Hep G2 cDNA library and the IGF-II and insulin subclones and Dr Anne Bentley (Sir William Dunn School of Pathology, Oxford, UK) for the genomic library. Christopher Graham is thanked for critical reading of the manuscript. This work was funded by the Cancer Research Campaign.

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