Some studies have suggested that insulin-like growth factor (IGF) pathway is related to premenopausal breast density, one of the strongest known breast cancer risk factors. This study was designed specifically to test the hypothesis that higher levels of IGF-I and lower levels of IGF-binding protein (IGFBP)-3 are associated with high mammographic breast density among premenopausal but not among postmenopausal women. A total of 783 premenopausal and 791 postmenopausal healthy women were recruited during screening mammography examinations. Blood samples were collected at the time of mammography, and plasma IGF-I and IGFBP-3 levels were measured by ELISA. Mammographic breast density was estimated using a computerassisted method. Spearman's partial correlation coefficients (r s ) were used to evaluate the associations. Adjusted mean breast density was assessed by joint levels of IGF-I and IGFBP-3 using generalized linear models. Among premenopausal women, high levels of IGF-I and low levels of IGFBP-3 were independently correlated with high breast density (r s = 0.083; P = 0.021 and r s = À0.124; P = 0.0005, respectively). Correlation of IGF-I with breast density was stronger among women in the lowest tertile of IGFBP-3 than among those in the highest tertile of IGFBP-3 (r s = 0.138; P = 0.027 and r s = À0.039; P = 0.530, respectively). In contrast, the correlation of IGFBP-3 with breast density was stronger among women in the highest tertile of IGF-I than among those in the lowest tertile of IGF-I (r s = À0.150; P = 0.016 and r s = À0.008; P = 0.904, respectively). Women in the combined top tertile of IGF-I and bottom tertile of IGFBP-3 had higher mean breast density than those in the combined bottom tertile of IGF-I and top tertile of IGFBP-3 (53.8% versus 40.9%; P = 0.014). No significant association was observed among postmenopausal women. Our findings confirm that IGF-I and IGFBP-3 are associated with breast density among premenopausal women. They provide additional support for the idea that, among premenopausal women, these growth factors may affect breast cancer risk, at least in part, through their influence on breast tissue morphology as reflected on mammogram.

Two 19-base-long oligonucleotides were synthesized, one complementary to the normal human j-globin gene (pA) and one complementary to the sickle cell (-globin gene (PS).The nonadecanucleotides were radioactively labeled and used as probes in DNA hybridization. Under appropriate hybridization conditions, these probes can be used to distinguish the pJA gene from the 3ss allele. The DNA from individuals homozygous for the normal fi-globin gene (fApA) only hybridized with the plA specific probe; the DNA from those homozygous for the sickle cell ,3-globin gene ((s.(s) only hybridized with the SsS specific probe. The DNA from heterozygous individuals (pATS) hybridized with both probes. This allele-specific hybridization behavior of oligonucleotides provides a general method for diagnosis of any genetic disease which involves a point mutation in the DNA sequence of a single-copy gene.

We have used oligonucleotide-directed mutagenesis to make a specific change in the f-lactamase (EC 3.5.2.6) (ampicillin resistance) gene of the plasmid pBR322. Evidence suggests that the active site for this enzyme may include a serine-threonine dyad (residues 70 and 71). By priming in vitro DNA synthesis with a chemically synthesized 16-base oligodeoxyribonucleotide, we have inverted the Ser-Thr dyad to Thr-Ser and thereby generated a mutant with an ampicillin-sensitive phenotype. This "double-mismatch" method is relatively simple and also very general because detection of mutants is at the level of DNA and involves only colony hybridization. Accordingly, the procedure can be applied to any DNA sequence and does not depend on the phenotype of the mutant.Rational study of the influence of amino acid sequence on the three-dimensional structure and function of a protein would benefit greatly from the ability to change specific residues. When the gene for the protein has been cloned and is expressed in an appropriate vector, site-directed mutagenesis (1, 2) provides a method for creating new proteins whose structural and functional characteristics may be ofpractical value or may offer significant mechanistic insights.Oligonucleotide-directed mutagenesis, the most specific form ofdirected mutagenesis, has been used to produce specific base changes in the single-stranded phage 4X174 (3, 4). The method ( Fig. 1) involves priming in vitro DNA synthesis with a chemically synthesized oligodeoxynucleotide that carries at least a single base mismatch with the complementary strand of the "wild-type" DNA. Because DNA polymerases require a double-stranded segment for initiation of DNA replication, the synthetic oligodeoxynucleotide primes DNA synthesis and is, itself, incorporated into the resulting heteroduplex molecule. After molecular cloning, semiconservative in vivo replication of this heteroduplex gives rise to homoduplexes whose sequences are either that of the original wild-type DNA or that of the synthetic oligodeoxynucleotide. With single-stranded circular DNAs, priming with oligodeoxynucleotides has been used to cause transitions, transversions, and deletions, in some cases very efficiently (5-11). Wallace et aL (12, 13) have extended the technique to double-stranded circular DNAs, and even though the frequency of directed mutagenesis may be lower, screening of colonies with the 32P-labeled synthetic oligodeoxynucleotide allows easy detection of the desired mutant (12, 13).The particular virtue of oligonucleotide-directed mutagenesis for structure-function studies lies in allowing one to produce mutant proteins with very specific changes in particular residues, which, for example, may be directly involved in catalysis or in determining substrate specificity. In this way, one can test ideas about the roles ofparticular amino acid side chains. Other procedures are valuable for producing random or less specific changes as, for example, in the creation of a mutant of f3lac-tamase (EC 3.5.2.6) with enhanced act...

This case-control study assesses relations of human papillomavirus (HPV) type 16 infection, sexual history, cigarette smoking, and oral contraceptive use to low- and high-grade cervical intraepithelial neoplasia (CIN). A total of 548 high-grade and 338 low-grade CIN cases and 612 controls were identified among women seen at a colposcopy clinic in Quebec, Quebec, Canada, in 1988-1989. Interviews, colposcopy, cervical scrapings, and colposcopically directed biopsies were performed. One pathologist reviewed all histologic slides. Southern blot techniques were used to assay specimens for HPV 16 DNA. Lifetime number of sexual partners was related to low- and high-grade CIN. Presence of HPV 16 DNA was associated with a 8.7-fold (95% confidence interval 5.1-15.0) elevation in estimated relative risk of high-grade CIN. Relative risk of high-grade CIN increased with amount of HPV 16 DNA (p < 0.0001). Estimated relative risk of high-grade CIN in current cigarette smokers was 2.4 (95% confidence interval 1.8-3.2) compared with never smokers and increased with number of pack-years of exposure (p < 0.0001). Long-term (6 years or more) users of oral contraceptives had an estimated relative risk of high-grade CIN of 1.9 (95% confidence interval 1.1-3.3) compared with those who never used such contraceptives. In contrast, presence of HPV 16 DNA, cigarette smoking, and oral contraceptive use showed little or no relation to low-grade CIN. Risk factors for low- and high-grade CIN may differ substantially.