The term "DNA fingerprint" has been used to describe the extensive restriction fragment length polymorphism associated with hypervariable minisatellites present in the human genome. Until now, it was necessary to hybridize Southern blots to specific probes cloned from human genomic DNA in order to obtain individual-specific restriction patterns. The present study describes the surprising finding that the insertfree, wild-type M13 bacteriophage detects hypervariable minisatellites in human and in animal DNA, provided no competitor DNA is used during hybridization. The effective sequence in M13 was traced to two dusters of 15-base pair repeats within the protein III gene of the bacteriophage. This unexpected use of M13 renders the DNA fingerprinting technology more readily available to molecular biology laboratories.E XTENSIVE RESTRICTION FRAGMENT length polymorphism (RFLP) associated with moderately repeated sequences has recently been described in the human genome (1-4). The hypervariable nature of these "minisatellites" has been exploited as a means of developing a powerfiul fingerprinting technique with applications in paternity testing, forensic medicine, and mapping of the human genome (5, 6). The probes used in such determinations were originally derived from segments of DNA discovered by chance within anonymous (3) or gene-associated pieces of genomic DNA (1, 4, 7). We report here that a sequence present in the protein III gene of the widely used M13 bacteriophage vector (8) allows detection of a distinct set of hypervariable minisatellites in human and animal DNA.In the course of a systematic search of RFLPs associated with the thyroglobulin gene, we observed that a set of probes corresponding to DNA segments subcloned in the 'bacteriophage M13 gave different results when used in the classical hybridization mixture [Denhardt's medium plus herring sperm DNA (9) tematically observed with the enzyme Hae III in the latter condition. It soon became evident that the pattern was unrelated to the nature of the insert and that wild-type M13 bacteriophages gave the same results. Nine unrelated individuals displayed different pattems while monozygotic twins were indistinguishable (Fig. 1, A and B). Similar blots hybridized with an M13 probe in the presence of herring sperm DNA showed no polymorphism (Fig. 1C). The logical explanation for this finding was that a segment in M13 hybridized to a hypervariable minisatellite that could be competed for by fish DNA. Ifthis were true,

No abstract

The availability of rat thyroglobulin cDNA clones was exploited to study the regulation of thyroglobulin gene transcription by thyrotropin (TSH). Groups of rats were subjected to treatments leading to reduction or increase in the rat serum TSH (rTSH) levels. Thyroid gland nuclei were isolated, incubated in vitro in the presence of 32P-labeled uridine triphosphate, and thyroglobulin transcripts were quantitated by hybridization to immobilized rat thyroglobulin cDNA clones. Transcription of the thyroglobulin gene was found to be very active in thyroid nuclei from control animals. It represented about 10% of total RNA polymerase II activity. Chronic hyperstimulation of the thyroid glands with endogenous rTSH was achieved in rats treated with the goitrogen propylthiouracil. No significant increase of thyroglobulin gene transcription could be measured in thyroid nuclei from these animals. On the contrary, a dramatic decrease in thyroglobulin gene transcription was observed in those animals in which endogenous rTSH levels had been suppressed by hypophysectomy or by the administration of triiodothyronine. Injection of exogenous bovine TSH in such animals readily restored transcriptional activity of the gene. Our results identify transcription as an important regulatory step involved in TSH action. They suggest that normal TSH levels induce close to maximal expression of the thyroglobulin gene but that continuous presence of TSH is required in order to maintain the gene in an activated state.

In a cell-free protein-synthesizing system from a rabbit reticulocyte lysate, total RNA extracted from cultured rat pituitary tumor (GH3) cells directed, in a dose-related manner, the synthesis of proteins that were precipitated by antisera specific to rat growth hormone (somatotropin) and rat prolactin. A marked decrease in growth hormone secretion and growth hormone mRNA activity was observed when cells were grown in a medium deficient in thyroid hormone. Addition of triiodothyronine in physiologic amounts both prevented and completely reversed this effect within 48 hr. Thyroid hormone had no effect on prolactin secretion or prolactin mRNA activity. These data suggest that thyroid hormone may stimulate synthesis of growth hormone throug induction of transcriptional activity. The possibility of an additional effect at the posttranscriptional level has not been excluded. Although thyroid hormone is believed to have a general effect on a variety of metabolic processes, some effects, at the molecular level, may be quite selective, as indicated by the observed changes in growth hormone but not prolactin mRNA activity. The GH3 cell model is useful in the study of triiodothyronine action because of independence from secondary hormonal effects caused by hypothyroidism and because simultaneous measurement of prolactin mRNA activity serves as a unique internal control. The demonstration of triiodothyronine (T3) binding to nuclear proteins raises the possibility that thyroid hormone may regulate gene expression. Earlier work from Tata's laboratory showed that thyroid hormone-induced protein synthesis was preceded by formation of new RNA (1). Later, DeGroot et al. and Dilman et al. showed increase in the poly(A)-rich fraction of RNA (2, 3). Demonstrations of stimulation of a specific mRNA by thyroid hormones were recently provided by Kurtz et al. (4) and by Roy et al. (5) for a2u-globulin in the rat. However, because a number of hormones are known to stimulate the synthesis of this protein (6) and because thyroid hormone profoundly alters the level of such hormones (7), experiments done in the whole animal do not provide sufficient evidence for the direct induction of -d2u-globulin mRNA by thyroid hormone. Samuels et al. have reported a quantitative correlation between nuclear T3 receptor occupancy and stimulation of growth hormone (GH, somatotropin) synthesis in cultured rat pituitary cells (8). We have adopted a similar experimental model to study more directly the action of thyroid hormone on the induction of a specific mRNA. In a recent report, we have shown that GH and prolactin mRNA activities in the total RNA extracted from a rat pituitary cell line (GH3) that actively synthesizes both hormones can be quantitated using a rabbit reticulocyte lysate cell-free system (9). In this report, we show that in the normal rat serum were 5.4 ,ug/100 ml and 60 ng/100 ml, respectively, and in the Tx rat serum were 0.6 ,g/100 ml and less than 20 ng/100 ml, respectively. RNA Extraction. Each RNA preparation was obta...

Complete absence of human somatomammotropin (hCS) was demonstrated in two patients experiencing an otherwise uneventful pregnancy. After delivery, DNA was prepared from the neonate blood or from the placenta and the integrity of the hCS-hGH gene cluster was investigated by Southern blotting and hybridization with an hCS cDNA probe. Patient 1 was found to be homozygous for a deletion involving hCS-A, hGH-V, and hCS-B. Patient 2 was a double heterozygote, with one chromosome bearing the same deletion as that of patient 1, while in the other, only the hCS-A gene was missing. Considerations relative to the frequency of the defect are derived from the present results.