Expression of alpha and beta integrins during terminal differentiation of cardiomyocytes.

BACKGROUND: In the myocardium, myocyte cell division is irreversibly blocked shortly after birth. The signal that initiates cell cycle withdrawal is unknown. The purpose of this study was to relate changes in expression of beta1 integrin and its associated alpha subunits to cardiomyocyte cell cycle progression during the fetal-to-neonatal developmental transition in rat. METHODS AND RESULTS: The developmental expression pattern and function of beta 1 integrin and several of its associated alpha subunits were examined using reverse transcription (RT) polymerase chain reaction (PCR) and beta 1 blocking antibodies. During the fetal to neonatal transition, a dramatic shift occurred in the levels of beta1 and alpha isoforms. At the 17-day fetal stage only beta 1A was present, which remained relatively constant until immediately after birth then decreased by 30% at the adult stage. By contrast, beta 1D appeared at fetal day 18, increased at neonatal day 2, and afterwards remained constant. This resulted in a ratio of beta 1A to beta 1D of about 1:1 in the adult heart. The integrin beta 1-associated subunits, alpha 3, alpha 6, and alpha 7, were expressed at extremely low levels in 17-day fetal cardiomyocytes. After birth alpha 3 and alpha 6 transiently increased at the 2-day neonatal stage, while alpha 7 isoforms B, C, and X2 progressively increased to the adult stage. Unlike skeletal muscle cells, fluorescence-activated cell sorting analysis (FACS) showed no down regulation of the alpha 5 beta 1 fibronectin receptor during cell cycle withdrawal. Treatment of cultured cardiomyocytes with beta1 blocking antibody inhibited the cell cycle in fetal but not in neonatal cells. CONCLUSION: These results suggest that progression through the cardiomyocyte cell cycle may be dependent upon cell attachment via integrin beta1 and correlate with changes that occur in beta1 spliced variants and their respective alpha isoforms.

Changes in E2F complexes containing retinoblastoma protein family members and increased cyclin-dependent kinase inhibitor activities during terminal differentiation of cardiomyocytes.

Cardiomyocyte terminal differentiation was examined by studying the interaction of retinoblastoma protein (pRb) family members with E2F during the developmental transition from 17-day fetal to 2-day neonatal. Additionally, the expression pattern of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors responsible for modulating the phosphorylation of pRb were studied. p107, pRb, and p130 are regulators of cellular proliferation, differentiation, and cell cycle exit and entry, respectively. The active, underphosphorylated form of these proteins targets the E2F family of transcriptional factors that play a critical role in the control of genes associated with DNA synthesis. Electromobility shift analyses demonstrated E2F complexed with p107 in proliferating fetal cardiomyocytes, whereas in 2-day neonatal cells, E2F was principally associated with p130 and a low level of pRb. At the 2-day neonatal stage, decreased protein levels were observed for cyclins D2, D3, and E, and CDK2 and CDK4. No changes were observed in the mRNA levels of the D-cyclins in neonatal cells; however, the transcripts for cyclins A and E and CDK4 were diminished. In skeletal myoblasts, differentiation is associated with induction of p21, a CDK inhibitor, by a MyoD-dependent pathway. Although heart cells lack MyoD, CDK assays demonstrated that the activity of CDKs 2, 4, and 6 were downregulated in 2-day neonatal cells, and CDC2 was increased. RT-PCR indicated that p21 mRNA was induced 1.4-, 2.0-, and 3.1-fold in the 2-day neonatal, 7-day neonatal, and adult stages, respectively, compared to the 17-day fetal stage. At the protein level, p21 also increased at the 2-day neonatal stage. Kinase inhibitory immunodepletion assays showed that CDK inhibitory activity was markedly increased in the 2-day neonate. Although mRNA levels of the p27 CDK inhibitor were unchanged, its protein level and inhibitory effect on CDK2 and CDK4 were increased. Thus, cardiomyocytes retain the capacity to proliferate until the early neonatal period when a series of changes occur, including a switch in pRb partners, a decrease in CDK levels and induction of CDK inhibitory activity, which is associated with terminal differentiation.

Characterization of cellular nucleic acid binding protein from Xenopus laevis: expression in all three germ layers during early development.

The Xenopus CNBP homologue (XCNBP) has been cloned from stage 14 neurula. XCNBP encodes a 18.4-kDa protein containing seven highly conserved zinc finger (Zn-finger) repeats (CX2CX4HX4CX2), with sequence similarity to human, mouse, rat, and yeast CNBP. A unique feature of XCNBP is that it contains a 10 amino acid (aa) deletion in the linker region between Zn-fingers 1 and 2, immediately downstream from an alternatively spliced exon of human CNBP isoforms. A similar deletion is found in mouse and yeast CNBP proteins. The deleted region lacks potential PEST and casein kinase II phosphorylation sites. Because CNBP proteins from a variety of species contain deletions in a similar region, these results suggest that the pattern of alternative processing of CNBP isoforms is highly conserved among metazoa and unicellular eukaryotes. XCNBP RNA is initially maternally derived and is widely expressed throughout early development at the gastrula, neurula, and tailbud stages. At the early gastrula stage, XCNBP is expressed in ectodermal, endodermal, and mesodermal germ layers. Previous data have demonstrated the presence of CNBP in the cytoplasm and nucleus. The interactions of CNBP with single-stranded DNA and RNA suggest that CNBP may serve dual functions in transcriptional and translational regulation in a wide variety of tissues during development.

Organization of the gene encoding cellular nucleic acid-binding protein.

The human cellular CNBP gene locus has been sequenced and is comprised of 6453 bp from the transcription start point (tsp) to the polyadenylation signal, and an additional 201 bp of 5' and 259 bp of 3' flanking sequences. The gene consists of five exons, four of which contain coding information for two alternatively spliced products, CNPB alpha and beta. The open reading frame (ORF) of 177 amino acids (aa) is encoded by exons 2 through 4 (CNBP alpha, M(r) 19463). The protein contains seven zinc-finger (Zf) domains. CNBP beta lacks seven aa in the linker region between the first two Zf, because of the use of an alternative 5' donor site within exon 2. The sixth Zf is the only Zf domain that is not completely encoded within a single exon and is interrupted by an intron (intron 4). The 5' untranslated region (UTR) contains 849 bp and is interrupted by intron 1. The 3' UTR, ending at the polyadenylation signal, is 857 bp long and is contained within exon 5. One Alu repeat was identified within intron 1, the largest intron of CNBP.

Alternatively processed isoforms of cellular nucleic acid-binding protein interact with a suppressor region of the human beta-myosin heavy chain gene.

Analysis of a series of human beta-myosin heavy chain (MHC) constructs with progressive deletions in the 5'-flanking region has localized a strong positive element at positions -298/277 with a repressor region located immediately upstream at -332/-300 (Flink, I. L., Edwards, J. G., Bahl, J. J., Liew, C.-C., Sole, M., and Morkin, E. (1992) J. Biol. Chem. 267, 9917-9924). A 49-base pair restriction fragment containing the suppressor element was used to screen a cardiac expression library. The 0.65-kilobase pair cDNA identified by this procedure was similar in sequence, except for the absence of a 21-base pair region encoding seven amino acids, to cellular nucleic acid-binding protein (CNBP), a 19-kDa zinc finger DNA-binding protein isolated earlier from liver, which may be involved in negative regulation of cholesterol biosynthesis (Rajavashisth, T. B., Taylor, A. K., Andalibi, A., Svenson, K. L., and Lusis, A. J. (1989) Science 245, 640-643). An additional clone identical to the one originally found in liver, referred to as CNBP alpha, was isolated from the cardiac library by hybridization screening. Gel mobility shift analysis indicated that CNBP alpha and CNBP beta isoforms preferentially interact with single-stranded DNA corresponding to the proximal and distal regions of the suppressor. When cotransfected with a beta-MHC reporter construct, CNBP alpha repressed activity in a dosage-dependent manner, whereas repression was not observed with the shorter construct (CNBP beta). Cotransfection of a combination of CNBP alpha and CNBP beta repressed reporter activity to an extent similar to cotransfection with CNBP alpha alone, suggesting that CNBP beta is not translationally active under these conditions. The results of RNase protection assays and genomic sequencing indicated that the alpha and beta isoforms are formed by alternative use of 5' donor sites within a single exon. These results suggest that CNBP isoforms may modulate the activity of the beta-MHC gene by interaction with a repressor region.

Thyroid hormone influences beta myosin heavy chain (beta MHC) expression.

3,5,3'-Triiodo-L-thyronine (T3) regulation of beta-MHC expression was studied in rat fetal heart cells using deletion mutants of both the rat and human promoter regions fused to a CAT expression vector. T3 inhibited the expression of human and rat beta-MHC constructs with an IC50 of about 1nM, which was similar to the EC50 for beta MHC-mRNA observed in cardiomyocytes. Deletion analysis of beta MHC promoter constructs suggested that a T3 response element (TRE) is located near the start of transcription. Possibly, T3-receptor binding at this site interferes with formation of the transcriptional initiation complex.

The structure of the human thyroxine binding globulin (TBG) gene.

A portion of the human X-chromosome (> 5 kb) encoding the translated portion of the thyroxine-binding globulin (TBG) gene was sequenced. The primary templates for sequencing were isolated from a human X-chromosome library (two positive plaques from 400,000 screened initially with a TBG cDNA probe) or were produced by PCR amplification using leucocyte genomic DNA as the amplification template. Potential hormone response elements (HREs) were identified at either end of the gene. These HREs have sequences based on the consensus half-site of thyroid hormone response elements, although it is unclear whether the structures are functional HREs. Other potential regulatory elements also were identified towards the 3' end of the gene.

Interleukin-6 inhibits corticosteroid-binding globulin synthesis by human hepatoblastoma-derived (Hep G2) cells.

Corticosteroid-binding globulin (CBG) belongs to the superfamily of serine proteinase inhibitors which include alpha 1-antitrypsin, alpha 1-antichymotrypsin, and T4-binding globulin. Interleukin-6 (IL-6), the main mediator of the acute phase phenomenon, increases alpha 1-antitrypsin and alpha 1-antichymotrypsin synthesis and decreases T4-binding globulin synthesis by human hepatoblastoma-derived (Hep G2) cells. This effect is predominantly at a transcriptional level. When Hep G2 cells were exposed to different concentrations of IL-6 for variable time intervals, IL-6 caused a dose- and time-dependent decrease in the amount of [35S]methionine-labeled CBG immunoprecipitated in the culture medium. This effect could be greatly reduced by preincubation of IL-6 with its neutralizing antibody and reversed by removing the cytokine from the culture medium. The secretion rate of CBG was not affected by cell exposure to IL-6. CBG mRNA steady state levels were reduced; changes in mRNA were quantitatively similar to changes in secreted protein. Nuclear run-off assays failed to show a change in the rate of transcription of the CBG gene. These data indicate that IL-6 diminishes CBG synthesis by Hep G2 cells acting at a posttranscriptional level, presumably through a reduced stability of mRNA. In view of the role of IL-6 in the inflammatory process and other acute phase phenomena, these data suggest that its effects on CBG synthesis might influence the bioavailability of cortisol indirectly and play a role in regulating the homeostatic process during these conditions.

Effects of interleukin-6 on the expression of thyroid hormone-binding protein genes in cultured human hepatoblastoma-derived (Hep G2) cells.

T4-binding globulin (TBG) shares a high degree of homology with two serpin antiproteases, alpha 1-antichymotrypsin (ACT) and alpha 1-antitrypsin (AT), whose synthesis is increased during the acute phase phenomenon, which accompanies trauma, infections, and neoplasms. Interleukin-6 (IL-6) is believed to be the main effector of the acute phase response. When evaluated in human hepatoblastoma-derived (Hep G2) cells exposed to different doses of the recombinant human cytokine for variable time intervals, IL-6 caused a dose- and time-dependent decrease in the secretion of [35S]methionine-labeled TBG, transthyretin (TTR), and albumin. The secretion of ACT and AT was increased. These changes were not due to alterations in the secretory process, since the kinetics of secretion of newly synthesized proteins were not modified. IL-6 did, however, cause a decrease in the steady state levels of mRNA for TTR, TBG, and albumin and an increase in ACT and AT mRNAs. In addition, nuclear run-off assay demonstrated a decrease in the transcription of TTR, TBG, and albumin genes and an increased transcription of the ACT gene. Quantitation of the results showed that changes in the secretion of proteins, in steady state mRNA levels, and in gene transcription were superimposable for each protein, indicating that IL-6 exerts its effect on thyroid hormone-binding proteins mostly at the transcriptional level and that TTR is the thyroid hormone-binding protein showing the most pronounced negative regulation by IL-6. The opposite effect of IL-6 on TBG and the antiproteases, despite their structural homology, underscores gene divergence among these proteins.

Characterization of a strong positive cis-acting element of the human beta-myosin heavy chain gene in fetal rat heart cells.

A strong positive element within the proximal promoter region of the human beta-myosin heavy chain (beta-MHC) gene that is required for high level expression in primary cultures of fetal rat heart cells was localized by transient assays and DNase I footprinting to positions- 277/-298. Using gel shift studies, this sequence was found to bind specifically at high affinity (Kd approximately 4 x 10(-9) M) to a transcriptional factor (beta F1) found in nuclear extracts from rabbit heart. Dimethyl sulfate interference studies suggested that beta F1 may bind as a dimer to two hexameric imperfect direct repeats containing the consensus sequence 5'-(C/G)-T-G-(T/A)-G-G-3'. Gel shift analyses suggested that beta F1 is related to the M-CAT factor, which is known to control muscle-specific expression of the cardiac troponin T gene. A clustered mutation of the region between the putative binding half-sites and within the "M-CAT"-like domain abolished beta-MHC promoter activity. The sequence of the positive element also contains binding motifs for several transcriptional factors that regulate viral and cellular genes, including AP4, AP5, TEF-1, and MyoD-like proteins. When multiple copies of the beta-MHC element were inserted downstream from the transcriptional initiation site of the thymidine kinase gene, it did not act as a classical enhancer, showing some dependence upon orientation.

A senescence up-regulated protein: the rat thyroxine-binding globulin (TBG).

Thyroxine-binding globulin (TBG), the major carrier of thyroid hormones in human serum, was thought to be absent in most species, including rodents. We demonstrated recently that in fact the rat possesses a TBG gene, virtually non-expressed in young adults, but actively transcribed during post-natal development. We now find that the TBG gene is also increasingly re-expressed during senescence. Evidence is presented suggesting that physiologically decreased thyroid hormone levels, characteristic of neonates and of ageing rats, might constitute a common factor inducing up-regulation of TBG in both developmental and ageing processes. Rat TBG is to our knowledge the first biochemical 'positive' (i.e. increasing) marker of non-pathological senescence, expressed at both biosynthetic and bloodstream levels.

Interaction of thyroid hormone receptors with strong and weak cis-acting elements in the human alpha-myosin heavy chain gene promoter.

The alpha-MHC gene is under positive regulation by 3,5,3'-triiodo-L-thyronine (T3), however, the mechanism by which T3 modulates its transcription is not clearly understood. We have used an avidin-biotin complex DNA binding assay and footprint analysis employing dimethyl sulfate interference and hydroxyl radical protection to characterize the interaction of T3 receptors with target sequences located in the 5'-flanking region of the human alpha-myosin heavy chain (MHC) gene. The results indicate that liver T3 receptors and in vitro transcribed-translated beta c-erbA bind with high affinity to a site (TRE1) located at positions -138/-158 base pairs upstream from the CAP site. The Kd of TRE1 for nuclear liver T3 receptors (0.82 nM) was less than that determined for the rat growth hormone gene (1.78 nM). An additional site (TRE2) located at positions -111/-129 was found which bound T3 receptors with considerably lower affinity (Kd = 23 nM). Methylation interference experiments demonstrated that T3 receptors interact with guanines, in TRE1 on the sense and antisense strands, within two octameric imperfect direct repeats (underlined) 5'-TCTGGAGGTGACAG-GAGGACA-3' (antisense strand sequence) containing the consensus sequence 5'-C(T/A)GGAGG(T/A)-3'. By contrast, methylation interference and hydroxyl radical footprinting demonstrate that the T3 binding element of TRE2 is not structurally similar to TRE1 except for a purine-rich octameric cluster (underlined, 5'-ATCAAAGGAGGAGGAGCCA-3') containing six guanines on the sense strand. These results suggest that differences in nucleotide sequences involved in T3 receptor-DNA complex formation determine the binding affinities of TRE1 and TRE2.

The hepatic biosynthesis of rat thyroxine binding globulin (TBG): demonstration, ontogenesis, and up-regulation in experimental hypothyroidism.

Using a human thyroxine binding globulin (TBG) cDNA probe, we demonstrate that rat liver contains two TBG mRNA species of different length, consisting of about 1.8 Kb and 2.4 Kb respectively. Slot blot analysis of the hepatic mRNAs from rats of different age reveals a fair correlation between the developmental trend of the messengers and that of the TBG circulating levels. Finally Northern blot and slot studies demonstrate that the increase of serum TBG induced in adults by thyroidectomy actually reflects an enhanced hepatic biosynthesis of the protein.

Sequence of the variant thyroxine-binding globulin of Australian aborigines. Only one of two amino acid replacements is responsible for its altered properties.

A form of thyroxine-binding globulin (TBG) with reduced affinity for hormone and increased susceptibility to heat and acid denaturation has been identified in Australian Aborigines (TBG-A). Results of heat denaturation of TBG established that the TBGA allele is X linked and has a frequency of 50.9% in Western Australian Aborigines. The sequence of an isolated TBGA allele differed at two positions from that of the normal TBG allele (TBGC). One substitution was in codon 191, ACA (threonine) rather than GCA (alanine), and the other was in codon 283, TTT (phenylalanine) instead of TTG (leucine). These nucleotide substitutions resulted in the loss of sites for the enzymes Bgl 1 and Tth 111 II, respectively. The nucleotide substitutions in the TBG-A allele was confirmed by digestion of genomic DNA segments amplified using the polymerase chain reaction. The Bgl 1 and Tth 111 II sites were absent in the genes of two Aboriginal men expressing TBG-A and were present in those of three Aboriginal and six Caucasian males expressing TBG-C. The TBG gene of a seventh Caucasian male possessed the Bgl 1 site but had lost the Tth 111 II site; sequencing of this allele revealed only the substitution in codon 283 identical to that in the TBGA allele. As the biochemical properties of TBGPhe-283 expressed by this individual were indistinguishable from normal TBGLeu-283, we believe that the abnormal properties of TBG-A are due to substitution of alanine for threonine at residue 191.

A mutation causing reduced biological activity and stability of thyroxine-binding globulin probably as a result of abnormal glycosylation of the molecule.

T4-binding globulin (TBG), a 54-kilodalton glycoprotein, is the major thyroid hormone transport protein in man. The exact nature of the mutations causing X chromosome-linked TBG deficiency, which affect about 1 in 2,500 newborn males, is unknown. Here we report the sequence of a unique variant TBG (TBG-Gary) encoding a protein with severely impaired T4 binding as well as decreased stability at 37 C, resulting in its rapid in vivo denaturation. A single nucleotide substitution in the codon for residue 96 of the mature protein replaces isoleucine with asparagine; this replacement creates an additional site for N-linked glycosylation. The anodal shift of TBG-Gary on isoelectric focusing gel electrophoresis suggests that this new site is likely glycosylated. Since glycosylated is required for TBG to assume its correct tertiary structure, but is not subsequently necessary for maintenance of the biological properties or stability of the molecule, we believe that the likely presence of additional carbohydrate probably affects a higher order structure of the molecule and is thus responsible for the reduced stability and hormone binding activity of TBG-Gary (TBGASN-96).

Detection of the thyroxine-binding globulin (TBG) gene in six unrelated families with complete TBG deficiency.

T4-binding globulin (TBG) is a glycoprotein of hepatic origin which transports thyroid hormone in serum. Inherited TBG defects in man are X-chromosome linked and are expressed in hemizygotes as complete deficiency, partial deficiency, or excess. Since TBG is not necessary for thyroid hormone action, affected subjects are healthy. Using DNA probes for human TBG, we searched for restriction fragment length polymorphisms in six affected males belonging to 6 unrelated families with inherited complete TBG deficiency and an equal number of normal males. TBG could not be detected in the serum of any of the TBG-deficient males by a specific and sensitive RIA capable of detecting as little as 5 micrograms TBG/L or 0.031% of the average normal serum TBG concentration. DNA isolated from white blood cells was digested with 11 restriction endonucleases, and the digests were submitted to DNA blot analysis using two cloned TBG-DNA probes which together covered the entire protein coding and the 5'-flanking sequences of the TBG gene. A total of 26 different bands were detected on DNA blots, identifying 18 restriction sites located within the 4.2-kilobase TBG gene, which includes intronic, exonic, and 5'-flanking sequences. This analysis, which sampled 2.3% of the total TBG genome, failed to reveal differences in fragment size among the 6 TBG-deficient and 6 normal males examined. One restriction endonuclease (NcoI) identified normal sequences at the putative promoter region of the gene, and four other endonucleases (TaqI, SstII, MspI, and HpaII) recognized the cytosine-guanine dinucleotide phosphate sequences representing potential mutation hot spots. Although C was methylated at these sites, no C to T (thymidine) transitions were found. These data suggest that large deletions, insertions, or rearrangements of the TBG gene, or mutations at sites of methylated cytosine-guanine dinucleotide phosphate dimers are not common mechanisms for inherited complete TBG deficiency in man.

Enzyme binding-inhibiting assay for iodothyronine 5'-monodeiodinase (5'-MD) and its application to isolation of complementary deoxyribonucleic acid clones for the 5'-MD in rat liver.

To identify and/or quantify type I T4 5'-monodeiodinase (5'-MD) immunologically, polyclonal antibodies were produced by immunization of rabbits with solubilized microsomal proteins (SMP) from rat liver. Pilot studies showed that the antibody binds to, but does not neutralize, rat liver enzyme. We have employed the polyclonal antibody to develop a 5'-MD enzyme binding-inhibiting assay (MBIA). For this purpose, active, inactive, or synthetic 5'-MD was preincubated with rabbit antibody and removed by Staphylococcus aureus protein-A (Staph-A). The 5'-MD-binding sites that were left on the Staph-A-bound rabbit antibody were assayed by adding active 5'-MD in fresh liver SMP. After centrifugation of Staph-A, the unbound 5'-MD enzyme activity was measured in the supernatant using [125I]rT3 in the presence of dithiothreitol. Incubation with 3.2 +/- 0.9 micrograms (mean +/- SEM; n = 4) rat liver SMP inhibited the binding of active 5'-MD to protein-A-bound antibody by 50%. Based on doses with similar 50% binding inhibitory activity, liver SMP were 2.3 times more potent than liver microsomes and 19 times more potent than liver homogenate; liver nuclei and mitochondria showed little or no inhibition of the binding of 5'-MD to antibody. The relative 5'-MD content of liver, kidney, pituitary, placenta, and cerebral cortex based on relative potency in the MBIA approximated 100:25:8:3:3. The threshold of the MBIA approximated 0.9 micrograms liver SMP/tube. The coefficient of variation approximated 2% within an assay and 6% between assays. To obtain a cDNA clone for 5'-MD we used the SMP antibody to screen a rat liver lambda gt11 cDNA expression library. Of 16 positive (antibody-reactive) clones that were isolated, the fusion proteins from only 2 (no. 23 and 54) inhibited the binding of 5'-MD in rat liver SMP to protein-A-bound rabbit antibody in a dose-dependent manner. Western blot analysis showed that the molecular size of liver protein encoded by clones 23 and 54 approximates 31K. Sequence analysis showed that clone 23 insert is 804 basepairs long, contains a single long open-reading frame, and is identical to clone 54. Southern blot analysis showed that clones 23 and 54 did not cross-hybridize DNA from other SMP antibody-positive clones. Northern blot analysis using clone 23 insert cDNA as the probe showed a hybridization band corresponding to a mRNA approximating 2.8 kilobases.(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of the human thyroxine-binding globulin gene to the long arm of the X chromosome (Xq21-22).

Thyroxine-binding globulin (TBG) is the major thyroid-hormone transport protein in the plasma of most vertebrate species. A recombinant phage (lambda cTBG8) containing a cDNA insert of human TBG recently has been described. With the cDNA insert from lambda cTBG8 used as a radiolabeled probe, DNA from a series of somatic-cell hybrids containing deletions of the X chromosome was analyzed by means of blot hybridization. The results indicated that the TBG gene is located in the midportion of the long arm of the X chromosome between bands Xq11 and Xq23. The gene then was mapped to band region Xq21-22 by means of in situ hybridization to metaphase chromosomes. Sequences on the X chromosome that are homologous to the cDNA probe are contained within a single EcoRI restriction fragment of 12.5 kb in human DNA. On the basis of the intensity of the hybridization signal on Southern blots, it was determined that the human TBG cDNA probe used in the present study shares significant homology with hamster and mouse sequences. A single EcoRI restriction fragment was recognized in both hamster (8.0-kb) and mouse (5.1-kb) DNA.