The beta-delta crossover leading to the beta delta hybrid gene of hemoglobin P-Nilotic is located within 54 base-pairs of the 5' end of exon 2 or between codons 31 and 50.

The crossover region of the beta delta hybrid gene of the hemoglobin variant Hb P-Nilotic was defined in detail through cloning and sequencing of appropriate DNA segments. The crossover must have occurred without loss of bases within a 54 base-pair stretch of DNA between bases 275 and 330 (or between amino acid residues 31 and 50), indicating that the exon 1 and IVS-1 originate from beta, and exon 2, IVS-2 and exon 3 from delta. The data support the speculation that the IVS-1, in contrast to IVS-2, has no effect on the expression of this hybrid gene.

Transient chloramphenicol acetyltransferase expression of the G gamma globin gene 5'-flanking regions containing substitutions of C----T at position -158, G----A at position -161, and T----A at position -175 in K562 cells.

The expression of G gamma is affected by mutations that occur in promoter sequences located in the 5'-flanking region of the gene. We have assayed the promoter activity of G gamma genes that have mutations of C----T at position -158 or G----A at position -161. In addition, we determined the activity of a promoter fragment containing T----A at position -175 (in the octamer motif) in combination with a -158 C----T which was produced during the polymerase chain reaction amplification procedure. Constructs containing these fragments were transfected by electroporation into K562 cells and the promoter activity was measured as chloramphenicol acetyltransferase activity. The data show a 4-5-fold enhancement of activity for the -158 C----T and the -161 G----A promoters over the 'normal' G gamma promoter and an 8-fold increase in the activity of the promoter with the double mutation (-158 C----T and -175 T----A). These results are consistent with data involving the increase in G gamma production in patients heterozygous for these mutations.

Staphylococcus aureus bacteremia among elderly vs younger adult patients: comparison of clinical features and mortality.

BACKGROUND: Previous studies give conflicting results regarding the effect of age on outcomes in Staphylococcus aureus bacteremia (SAB). These studies have been limited by retrospective design or small sample size. METHODS: We conducted a prospective cohort study of 385 patients with SAB aged 18 to 90 years. The setting was a large academic medical center. We observed patients from diagnosis of SAB to discharge or death. Discharged patients were contacted 12 weeks after their first positive culture findings. Data were collected on demographics, comorbid conditions, focus of infection, length of stay, and outcome. Primary outcomes were total mortality and death due to SAB. RESULTS: Comparisons were made between 145 patients, aged 66 to 90 years, and 240 patients, aged 18 to 60 years. Forty-three (29.7%) of the elderly patients and 36 (15%) of the younger patients died. Death directly attributable to SAB occurred in 21 (14.5%) older and 15 (6.3%) younger patients. After adjusting for confounding variables, older patients continued to have higher total mortality (odds ratio, 2.21; 95% confidence interval, 1.32-3.70), and higher mortality from SAB (odds ratio, 2.30; 95% confidence interval, 1.13-4.69). Infection with methicillin-resistant S aureus was associated with higher total mortality in the elderly (odds ratio, 2.59; 95% confidence interval, 1.23-5.43). CONCLUSIONS: Staphylococcus aureus bacteremia among the elderly is associated with high mortality. Both total mortality and mortality directly attributable to SAB are more than twice as likely in older patients. Infection with methicillin-resistant S aureus carries a worse prognosis than infection with methicillin-sensitive S aureus in the elderly.

Synthesis of a fixed-length single-stranded DNA probe by blocking primer extension in bacteriophage M13.

A simple and efficient technique has been developed for preparing radiolabeled single-stranded (ss) probes of determined length and high specific radioactivity. The human beta-globin gene intervening segment II (IVSII) fragment (0.9-kb) was inserted between the EcoRI and BamHI sites of M13mp11 and used as a template for ss probe synthesis. The M13 hybridization probe primer (M13 Hpp) was annealed to the recombinant M13mp11-beta IVSII template DNA. This M13 Hpp was next blocked by the enzymatic addition of a dideoxy adenosine monophosphate (ddAMP) residue to the 3' OH group of the primer. The M13 universal sequencing primer was then annealed and used to prepare an ss copy of the beta-IVSII fragment. Synthesis of the ss fragment was terminated by the presence of the dd-blocked M13 Hpp yielding a specific 0.9-kb ss beta-IVSII probe.

Identification of restriction-fragment length polymorphisms for the human chorionic gonadotropin-beta/luteinizing hormone-beta gene cluster.

OBJECTIVE: To determine whether restriction fragment length polymorphisms are present using a deoxyribonucleic acid (DNA) probe for human luteinizing hormone beta subunit (hLH-beta). If the gene for hLH-beta is polymorphic, genetic diagnosis of disorders of luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) production could become possible. DESIGN: Study of genomic DNA from controls with a variety of restriction enzymes to identify polymorphisms. SETTING: Laboratories of the Department of Obstetrics and Gynecology, Department of Oral Biology, Medical College of Georgia, Augusta, Georgia. PATIENTS: Unrelated control men and women seen in clinics at the Medical College of Georgia. INTERVENTIONS: Genomic DNA was extracted from patients and digested with eight different restriction enzymes for the study of the hLH-beta gene by Southern analysis. MAIN OUTCOME MEASURE: Fragment (band) sizes on radiographs from Southern blots were compared with those from molecular weight standards. CONCLUSIONS: Restriction fragment length polymorphisms were identified for four of the restriction enzymes, DraI, HincII, MboI, and KpnI. These polymorphisms may be useful in the diagnosis of disorders of hLH and hCG production.

Gonadotropin-releasing hormone, follicle-stimulating hormone beta, luteinizing hormone beta gene structure in idiopathic hypogonadotropic hypogonadism.

OBJECTIVE: To determine if the genes for gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone beta (FSH beta), and luteinizing hormone beta (LH beta) are present, and if so, whether gene structure is normal in patients with idiopathic hypogonadotropic hypogonadism (IHH). DESIGN: Patients with clinical and laboratory characteristics of IHH were studied at the deoxyribonucleic acid (DNA) level to assess gene structure. SETTING: This study took place in an academic setting. PATIENTS: Human volunteers with documented IHH and fertile controls were studied. INTERVENTIONS: Genomic DNAs were extracted from each patient, Southern blots were constructed and hybridized to DNA probes for GnRH, FSH beta, and LH beta. DNA samples were also subjected to polymerase chain reaction analysis. MAIN OUTCOME MEASURES: Gene structure was assessed by analysis of autoradiographs and gel electrophoresis of polymerase chain reaction products in both the study patients and controls. RESULTS: Each analysis for FSH beta, LH beta, and GnRH demonstrated the same sized fragments in both the study group and control group. A 1.2-kilobase fragment containing the coding region for GnRH was present in all patients with IHH and controls by polymerase chain reaction. CONCLUSIONS: The genes for GnRH, LH beta, and FSH beta are present in patients with IHH. No large deletions or rearrangements of any of these genes were identified in any of these patients.

The formation of poly A-containing RNA in rat liver after administration of 3-methylcholanthrene.

The incorporation of [32P] orthophosphate into liver poly A-rich cytoplasmic RNA was investigated in rats which were pretreated with 3-methyl cholanthrene (3MC) or with the vehicle, corn oil. This incorporation was markedly increased within 6 h after administration of the polycyclic hydrocarbon and reached a maximum by 24 h, i.e., 33-fold increase. The poly A-rich RNA fraction was examined by gel electrophoresis and the specific activity of individual species was shown to be elevated.

Effects of estradiol on early messenger RNA in male Xenopus laevis liver.

Cytoplasmic RNA isolated from male Xenopus liver between 1 and 6 h of hormone treatment was separated into distinct classes by Sepharose 4B chromatography. Assay of the mRNA activity of these RNA species in the wheat germ cell-free system demonstrated 2 populations of mRNA activity (peak 2 and peak 3). The activity of 1 population of mRNA (peak 2) was inducible by estradiol while the other (peak 3) remained unaltered for at least 6 h. mRNAs obtained by Sepharose 4B chromatography were also translated in the Xenopus oocyte. The translation products were analyzed by Sephadex chromatography and SDS-gel electrophoresis.

Translation of hormone-induced messenger RNA in amphibian oocytes: I. Induction by estrogen of messenger RNA encoded for vitellogenic protein in the liver of the male African clawed toad (Xenopus laevis).

Induction of the synthesis of the vitellogenic proteins, lipovitellin and phosvitin, in the liver of the male African clawed toad (Xenopus laevis) was investigated as a function of time after treatment with estradiol-17beta [1,3,5(10)-estratriene-3,17beta-diol]. The appearance of mRNAs encoded for lipovitellin and phosvitin in the cytoplasmic fraction of the liver was assayed by microinjections of hepatic mRNA preparation [either polyribosomes or poly(A)-rich RNA] into oocytes obtained from mature female toads. Oocytes were then incubated in the presence of radioactive amino acid(s) at 19 degrees for periods of time varying from 4 to 18 hr after microinjection. The results show that at 2 hr after hormone treatment more mRNA was present in the cytoplasm, and that from 2 to 72 hr after treatment the level of induced mRNA increased almost linearly to 110% above the control values. Experiments employing specific lipovitellin antiserum indicated no radioactive lipovitellin among the proteins synthesized in oocytes microinjected with hepatic mRNAs isolated from 3 to 9 hr after hormone treatment. However, a marked synthesis of immunoprecipitable, radioactive lipovitellin and an enhanced incorporation of [3H]serine occurred in the oocytes microinjected with hepatic mRNA preparations obtained from toads treated with hormone for 12 or more hr. The identities of the proteins encoded by the mRNAs induced early in estrogen action (2-9 hr) in the male amphibian liver are unknown. It is surmised that some of these proteins may function in the regulation of the subsequent synthesis of the vitellogenic proteins.

Effect of calmodulin inhibitor, Stelazine, on the endocytosis of vitellogenin and transglutaminase activity in Xenopus laevis oöcytes.

The specific endocytosis of vitellogenin was measured in the presence of various concentrations of Stelazine, a specific inhibitor of the calcium regulating protein, calmodulin. Stelazine (200 microM) was found to inhibit the endocytosis of vitellogenin by 63% as determined by decreased uptake of vitellogenin into transitional yolk bodies and yolk platelets. The enzymatic activity of transglutaminase, a calcium-requiring enzyme implicated in receptor-mediated endocytosis, was not affected by these concentrations of Stelazine.

Assignment of the human 2',3'-cyclic nucleotide 3'-phosphohydrolase gene to chromosome 17.

2',3'-Cyclic nucleotide 3'-phosphohydrolase (CNP) has been used as a general oligodendrocyte and Schwann cell marker enzyme within the nervous system and has been the intense target of a number of recent studies. In this report, we determined the chromosomal localization of the human CNP gene using PCR on two somatic cell DNA panels. PCR amplification, using four primer pairs across an intron, confirms that the CNP gene is localized to chromosome 17. We also present the complete intron sequence of the human gene usd to make the assignment. This intron contains a c----t polymorphism located at nucleotide 1215, which may be of use in mapping the CNPase gene more precisely within chromosome 17.

Estrogen receptor messenger ribonucleic acid changes during Leydig cell development.

Mature (60-65 days old) male Sprague-Dawley rats received a single i.p. injection of ethane dimethane sulfonate (EDS, 100 mg/kg BW) and were killed at different times from Days 2 to 60 posttreatment. Bands of cells enriched in precursor Leydig cells (PLCs) and Leydig cells (LCs) were isolated from the testis of EDS-treated rats and age-matched controls using a collagenase digestion-Percoll gradient method. Total RNA extracted from the PLC and LC fractions was subjected to reverse transcriptase polymerase chain reaction (RT-PCR) to detect estrogen receptor (ER) mRNA. The RT-PCR results demonstrated that ER mRNA was present in both LC and PLC fractions. Quantitative RT-PCR analysis, using rabbit beta-globin mRNA as the internal standard, showed that ER mRNA in the PLC fraction was 20-fold higher than in the LC fraction in control testis. After EDS treatment, ER mRNA levels in the PLC fraction decreased and reached a nadir at Day 16 posttreatment. Thereafter, ER mRNA in the PLC fraction gradually increased and returned to control PLC levels. In contrast, ER mRNA levels in the LC fraction in controls and at Days 16-45 posttreatment remained constant. To correlate the changes in ER mRNA levels with LC differentiation, in vitro testosterone (T) production by PLC- and LC-enriched fractions in the presence or absence of 50 mIU hCG was measured by RIA. T production in the control PLC fraction was low (1/10th that in the control LC fraction), and hCG addition resulted in only a 1.5-fold stimulation (relative to a 7.5-fold stimulation in LCs). In the PLC fraction, T production was not detectable at Days 2 and 10 after EDS treatments, began to respond to hCG stimulation with increased T production at Day 16, and reached a maximum between 4 and 6 wk after EDS treatment. By Day 60 posttreatment, T production in the PLC fraction decreased and returned to control PLC levels. Testosterone production in the LC fraction was not detectable at Days 2 and 10 posttreatment. From Days 16 to 60 posttreatment, LC basal and hCG-responsive T production increased gradually and returned to control LC levels. It is concluded that functional LCs are regenerated from the PLCs and that both these cell types possess ER mRNA. It is interesting to note that PLCs exhibit higher levels of ER mRNA than do LCs. A decrease in ER mRNA in PLCs appears to coincide with the early differentiation process to yield LCs. Thus, estradiol-17 beta produced locally in the testis by the LCs might act via its receptor as a paracrine substance to impede PLC development into LCs. It is therefore possible that either a decrease in E2 production or a decrease in ER and its mRNA in PLCs would then release the PLCs to begin the regeneration process.

Two mutations in the locus control region hypersensitivity site-2 (5' HS-2) of haplotype 19 beta s chromosomes alter binding of trans-acting factors.

There are five major haplotypes associated with sickle cell anemia (SS). Individuals homozygous for haplotypes 3 (Senegal) and 31 (Saudi Arabian) have high fetal hemoglobin (HbF) levels (15 to 30% of total hemoglobin) whereas individuals homozygous for haplotypes 17 (Cameroon), 19 (Benin), and 20 (Bantu) have low HbF levels (1 to 10%). We previously identified several point mutations in the LCR 5'HS-2 that were specific for haplotype 19 beta s chromosomes (compared to the GenBank HUMHBB reference sequence, T-->G at position 8580, A-->G at position 8598, and A-->T at position 9114). We postulated that one or more of these mutations may alter the binding of specific trans-acting factors and ultimately affect the expression of HbF in these sickle cell patients. We performed gel mobility shift assays using 32P-end-labeled double-stranded 19mers corresponding to each of the LCR 5'HS-2 normal (GenBank) and mutant sequences. Nuclear extracts prepared from HeLa and HEL cells were used in our experiments and neither the normal nor mutant sequence at position 8580 bound trans-acting factors in either nuclear extract. The 8598 mutant increased binding of Sp1; using purified protein and both nuclear extracts. HEL extracts were used to quantify the increase in Sp1 binding to the 8598 mutation and we found an increase in binding of 66 and 47%, respectively, in two shifted bands. The 9114 mutation sharply decreased binding of an unknown trans-acting factor by 74%. This factor was present in both HeLa and HEL nuclear extracts.

Effects of estradiol-17beta in the male Xenopus laevis: isolation and translation of cytoplasmic messenger RNA populations.

Total Xenopus liver cytoplasmic RNA isolated following long-term estrogen administration (14 days) was fractioned using Sepharose 4B chromatography. One of the Sepharose 4B peaks was shown to contain RNA with a molecular weight reported for vitellogenin mRNA (approximately 34S). The presence of estrogen-induced vitellogenin mRNA in the peak 5 RNA was determined by translation of the RNA in the oocyte and analysis of the oocyte translational products by immunoprecipitation with anti-vitellogenin. Sepharose 4B peaks 2 and 3 were also observed to contain estrogen induced mRNA populations sedimenting between 9--18S. These findings suggest that Sepharose 4B chromatography might prove useful in separating different mRNA populations following estrogen-induced gene activation.

Sequence variations in the 5' flanking and IVS-II regions of the G gamma- and A gamma-globin genes of beta S chromosomes with five different haplotypes.

We have amplified and sequenced the 5' flanking and the second intervening sequence (IVS-II) regions of both the G gamma- and A gamma-globin genes of the beta S chromosomes from sickle cell anemia (SS) patients with homozygosities for five different haplotypes. The sequencing data, compared with previously published sequences for the normal chromosomes A and B, show many similarities to chromosome B for haplotypes 19, 20, and 17, while haplotypes 3 and 31 are remarkably similar to chromosome A and also similar to each other. Several unique mutations were found in the 5' flanking regions (G gamma and A gamma) of haplotypes 19 and 20 and in the IVS-II segments of the same genes of haplotypes 19, 20, and 17; the IVS-II of haplotypes 3 and 31 were identical to those of chromosome A. Dot-blot analyses of amplified DNA from additional SS patients with specific probes have confirmed that these mutations are unique for each haplotype. The two general patterns that have been observed among the five haplotypes have most probably arisen by gene conversion events between the A and B type chromosomes in the African population. These patterns correlate with high and low fetal hemoglobin expression, and it is speculated that these and other yet unknown gene conversions may contribute to the variations in hemoglobin F and G gamma levels observed among SS patients. In vitro expression experiments involving the approximately 1.3-kb 5' flanking regions of the G gamma- and A gamma-globin genes of the beta S chromosomes with the five different haplotypes failed to detect differences between the levels of expression, suggesting that the sequence variations observed between these segments of DNA are not the primary cause of the differences in hemoglobin F levels among the SS patients.

Isolation and characterization of the nuclear matrix from the male Xenopus laevis following estrogen administration: kinetics of [3H] uridine incorporation.

At various times following estrogen administration, the nuclear matrix was isolated from the liver of male Xenopus laevis by sucrose gradient centrifugation of nuclei treated with a high-salt buffer and DNase I in the presence of a proteolytic inhibitor (PMSC--phenylmethyl sulfonyl chloride). Electron micrographs of the nuclear matrix demonstrate a sponge-like network attached to a well-defined inner envelope with a ribosome-free outer envelope. Chemical analyses show that the HSB-DNase-treated nuclei consist of 16% DNA, 2% RNA, and 82% protein, a composition that is consistent with that of nuclear matrices isolated from other species. The specific activity of the matrix-associated RNA following estrogen treatment appears to be maximally enhanced after 5 h and decreases until approximately 12 h, when the activity begins to increase again.

Inhibition of gene expression by the Ggamma 5' flanking region of the Bantu beta(s) chromosome.

Beta(s)-chromosome haplotypes are peculiar to specific regions of Africa and Asia and are associated with the occurrence of different fetal hemoglobin (Hb) levels in sickle cell patients. Among these haplotypes, beta(s)-chromosomes found in the Senegal and the Arab-India regions are associated with relatively high levels of HbF expression, whereas those around the Benin, Bantu, and the Cameroon regions show low levels of HbF expression. The roles of 5'HS2 and the 5' flanking (promoter region) region in the expression of globin genes are well documented. Haplotype specific variations are found in these regions and have been postulated to be involved in the regulation of HbF expression. In this study, we have analyzed the effect of sequence variations in regulatory regions of the Bantu 5'HS2 and 5' flanking region of the Ggamma gene on CAT expression. A diminution was observed in K562 cells when the promoter originated from the Bantu beta(s) chromosome. The decreased expression was independent of the origin of the 5'HS2 sequence--combinations of the Bantu promoter were measured with the Benin, Bantu, or Senegal 5'HS2 sequences in K562 cells. However, expression of the same plasmids in murine erythroleukemic (MEL) cells showed no difference in CAT expression among the various sequence combinations studied.

A chromosome with five gamma-globin genes.

Globin gene mapping of DNA from a Black newborn resulted in the detection of a chromosome with five gamma-globin genes. Based on results from digests with enzymes EcoRI and PstI, we concluded that the three genes between the 5'G gamma and 3'A gamma genes are G gamma genes with a possible 5' segment derived from A gamma. The high G gamma level in the fetal hemoglobin (Hb F) of the baby is consistent with this view. Family relationships were such that speculation as to the mechanism causing this quintuplication of the gamma-globin genes was not possible.

The effect of alpha-thalassemia on the level of hybrid hemoglobin variants in heterozygotes.

The influence of a relative deficiency in alpha chain production on the amount of Hemoglobins Kenya, P-Nilotic, and Lepore was determined. The level of these hybrid hemoglobins in heterozygotes was correlated to various states of alpha chain deficiency by: 1) quantitation of the variants in blood samples and comparing these data with the number of alpha globin genes determined by gene mapping, 2) in vitro recombination experiments involving isolated non-alpha chains and normal alpha chains, and 3) in vitro heat stability analyses of the isolated hemoglobins. Hb Kenya, composed of normal alpha and gamma-beta hybrid chains, is heat labile, has a decreased ability to combine with alpha chains, and its level in heterozygotes is greatly decreased when a concomitant alpha-chain deficiency (alpha-thalassemia) is present. Such a posttranslational control mechanism was not observed for Hb Lepore, with normal alpha chains and delta-beta hybrid chains, and Hb P-Nilotic, with normal alpha chains and beta-delta hybrid chains. The latter two variants are heat stable, and their hybrid chains combine equally well as normal beta chains with normal alpha chains. Hb P-Nilotic is more heat stable than Hb A and its in vitro formation is increased over that of Hb S, and perhaps even Hb A, in conditions of severe alpha chain deficiency.

Identical nucleotide sequences of the 3'A gamma globin gene enhancer elements from four different chromosomes.

We have determined the nucleotide sequence of the 2,360-bp long EcoRI fragment from four chromosomes; this fragment is located 3' to the A gamma globin gene and is considered to contain the enhancer element identified by Bodine and Ley. The chromosomes were from an Arabian sickle cell anemia patient with high Hb F and a homozygosity for haplotype No 31 and from a black sickle cell anemia patient with low Hb F and a homozygosity for haplotype No 19. A third chromosome carried the determinant for a nondeletional hereditary persistence of fetal hemoglobin seen in a Chinese subject, and the fourth was a normal chromosome from a Yugoslavian subject. Twenty-one differences were observed when a comparison was made with the published sequence; no differences were seen between the sequences of the four different samples except for an additional mutation in the Chinese. These data make it unlikely that specific mutations within this sequence are associated with increases in G gamma and A gamma production.