Allelic exclusion in transgenic mice that express the membrane form of immunoglobulin mu.

Antibody-producing cells display a special form of regulation whereby each cell produces immunoglobulin from only one of its two sets of antibody genes. This phenomenon, called allelic exclusion, is thought to be mediated by the product of one heavy chain allele restricting the expression of the other. Heavy chains are synthesized in two molecular forms, secreted and membrane bound. In order to determine whether it is specifically the membrane-bound form of the immunoglobulin M (IgM) heavy chain (mu) that mediates this regulation, transgenic mice were created that carry a human mu chain gene altered so that it can only direct the synthesis of the membrane-bound protein. The membrane-bound form of the human mu chain was made by most of the B cells in these animals as measured by assays of messenger RNA and surface immunoglobulins. Further, the many B cells that express the human gene do not express endogenous mouse IgM, and the few B cells that express endogenous mouse mu do not express the transgene. Thus, the membrane-bound form of the mu chain is sufficient to mediate allelic exclusion. In addition, the molecular structures recognized for this purpose are conserved between human and mouse systems.

Discordant effects of aging on prolactin and luteinizing hormone-beta messenger ribonucleic acid levels in the female rat.

To examine the molecular genetic basis for the age-related increase in PRL secretion and decrease in LH production in the rat, we measured steady state levels of PRL and LH beta mRNA in pituitary homogenates and cell lysates from monolayer adenohypophyseal cultures. These mRNA levels were compared with the corresponding levels of immunoreactive PRL and LH in sera and culture media. Paired groups (n = 4-10/group) of intact and 4-week ovariectomized mature (6-7 months old) and old (23-25 months old) female Wistar rats were studied. Serum PRL levels were 550% higher in intact old vs. mature rats (P less than 0.001), whereas the corresponding pituitary homogenate levels of PRL mRNA were similar (P greater than 0.4). Medium PRL concentrations were 230% greater (P less than 0.006) whereas cell lysate concentrations of PRL mRNA were unaltered (P greater than 0.2) in monolayer cultures from intact old vs. mature rats. Serum PRL levels were 650% higher (P less than 0.003) and pituitary homogenate PRL mRNA levels were slightly increased (P less than 0.04) in ovariectomized old vs. mature rats. Neither serum LH values (P greater than 0.07) nor pituitary homogenate LH beta mRNA levels (P greater than 0.1) differed in intact old and mature rats, whereas the corresponding medium concentrations of LH were reduced (P less than 0.001). Ovariectomized old vs. mature rats exhibited reductions in serum (P less than 0.02) and medium (P less than 0.001) LH concentrations, as well as in pituitary homogenate (P less than 0.002) and cell lysate (P less than 0.006) LH beta mRNA levels. Thus, these data revealed coordinate decreases with age in LH beta mRNA and LH secretion, particularly in ovariectomized rats, suggesting an age-related alteration at or before LH beta gene transcription. These findings parallel observations on other genes whose products change with age. In contrast, the observation that the increased secretion of PRL in old rats is accompanied by little or no increase in PRL mRNA is novel and suggests that age-related alterations in PRL gene expression proceed through a posttranscriptional mechanism.

The lac operator as a phenotypic label for DNA fragments cloned in Escherichia coli.

To confer a detectable phenotype on any DNA fragment cloned in Escherichia coli, one can label the fragment by ligating it to the lac operator so that host cells can be identified as blue colonies on agar plates. This screening strategy is similar to that used for the pUC and M13 series of vectors, but does not require the vector to contain the lacZ gene. Instead, the presence of the lac operator on the multicopy vector results in the induction of the host cell beta-galactosidase by titrating out the repressor. This paper describes how pUC/M13 vectors or synthetic oligodeoxynucleotides can be used to supply the operator label, and shows how this method has been used to position unique restriction sites for initiating BAL 31 deletions. This approach may be particularly helpful when a given DNA fragment is to be cloned in many different constructs or is to pass through many sequential cloning steps.

Plasmid cloning vectors resistant to ampicillin and tetracycline which can replicate in both E. coli and Haemophilus cells.

We have constructed two plasmid vectors, pHCV5 and pHVTl, which will replicate both in Haemophilus and in Escherichia coli. Both contain the ampicillin-resistance gene and the replication origin from a Haemophilus plasmid, pRSF0885. Both also contain the pBR322 origin and therefore can be amplified in E. coli by chloramphenicol treatment. The plasmid pHCV5 contains the tetracycline-resistance gene of pBR322, and pHVT1 contains the analogous region from the transposon Tn10.

An eleven-base-pair sequence determines the specificity of DNA uptake in Haemophilus transformation.

Only certain DNA fragments are taken up efficiently by component Haemophilus cells; this implies that efficient uptake requires the presence of a specific nucleotide sequence on the incoming DNA (Sisco and Smith, 1979). To determine the structure of this "uptake site", we have isolated and sequenced four small fragments of cloned H. parainfluenzae DNA which retain the ability to be taken up by cells. These fragments have a sequence of eleven base pairs in common, 5'-AAGTGCGGTCA-3' and ethylation of certain phosphoryl groups in this sequence causes significant decreases in fragment uptake. We conclude that this is the sequence of the uptake site.

Regions of evolutionary conservation between the rat and human prohibitin-encoding genes.

We have analyzed and compared the 5' promoter region, the intron structure and the exon-intron flanking sequences in the rat and human prohibitin-encoding genes (PHB). Comparative analysis of a 350-nt region immediately 5' to and including the first exon identifies eight highly conserved regions, four of which correspond to binding sites for known transcriptional control proteins (CCAAT box, 'SV40' site and two Sp1 sites). The promoter lacks a TATA box. Four transcription start points (tsp) clustered within a 35-bp region were identified by rapid amplification of cDNA ends (RACE). The exon-intron boundaries in rat and human are highly conserved, with identical positioning of splice junctions. PCR analysis with conserved exon primers was used to detect length variation between rat and human PHB, and length differences were observed in all of the introns.

The proliferation theory of rejuvenation.

A theory concerning the molecular basis of rejuvenation is presented that postulates a central role for cell proliferation. This theory assumes that aging is due to the accumulation of multiple forms of molecular damage and that rejuvenation is due to repair. The advantages of proliferation as a means of repair are described and it is proposed that cell proliferation is required for full rejuvenation. This proliferation theory offers several advantages: a different perspective on the question of which organisms age; an explanation of aging-related phenomena that are not well handled by traditional aging theories; a novel approach to altering the aging rate; and testable implications for the design of new experimental systems and therapeutic interventions.

Isolation and identification of aging-related cDNAs in the mouse.

To identify genes whose expression changes as a function of aging, we screened mouse cDNA libraries with cDNAs from mice of different ages. Specifically, whole-mouse cDNA libraries were constructed in lambda gt10 using poly(A) RNA from young (3 month) and old (27 month) C57BL/6J inbred mice and these lambda plaques were hybridized with radioactive cDNAs made from pooled poly(A) RNA from animals 3 or 33 months of age. Five clones were isolated that showed an aging-related pattern of expression and four of these were identified by computerized sequence matching to the GenBank database: MUP2 (a major urinary protein); Q10 of the MHC locus; a cytoskeletal actin gene; and creatine kinase. One gene whose expression increases with aging and is most abundant in spleen remains unidentified. All five cDNAs showed 4-fold to 17-fold changes with aging in their steady-state mRNA levels in at least one tissue.

Aberrant gene expression and aging: examination of tissue-specific mRNAs in young and old rats.

It has been suggested that aberrant gene expression may play a role in aging. To test this possibility, we examined the steady-state mRNA levels for five tissue-specific genes of known function in young (6 month) and old (24 month) rats. Six different tissues from three animals of each age were analyzed using a hybridization assay estimated to be able to detect one mRNA copy per cell. At this level of sensitivity, no aberrant gene expression was seen. The results indicate that these tissue-specific genes retain their fidelity of expression with age.

Approach to the isolation of antiproliferative genes.

Poly(A) RNAs from normal rat liver and senescent human fibroblasts appear to have more antiproliferative activity than RNAs from regenerating rat liver and early passage human fibroblasts. We have screened two rat liver and one human liver library by differential hybridization and isolated four candidate cDNAs for this antiproliferative activity; one is fibronectin and three others do not match to any sequence in the mammalian portion of the GENBANK database. We are currently testing the antiproliferative nature of these cDNAs by microinjection of hybrid-selected RNA, and we describe an alternative strategy for cloning such genes based on construction of a cDNA library in an RNA expression vector.

DNA damage and repair with age in individual human lymphocytes.

Previous biochemical studies on DNA repair competence and aging have been limited to techniques, such as alkaline elution or nucleoid sedimentation, involving mass cell populations. These techniques provide no information about the distribution of DNA damage and repair among individual cells and are unlikely to detect age-dependent changes affecting a minor fraction of the cell population. We have recently described a microgel electrophoretic assay (Singh et al., 1988) that measures, at the level of the individual cell, single-strand DNA breaks and alkali-sensitive sites. Here, we employ this method to analyze DNA damage and repair in lymphocytes isolated from the peripheral blood of 31 subjects (23 males and 8 females aged 25-91 years) and exposed in vitro to 200 rads of X-irradiation. While basal (pre-irradiation) levels of damage were independent of the age of the donor, an age-dependent increase in DNA damage was observed immediately following irradiation. For all subjects, the mean level of DNA damage was restored to pre-irradiation control levels within 2 h of incubation at 37 degrees C. However, a distribution analysis of DNA damage among cells within each sample indicated the presence of a few highly damaged cells (4-16%) in the 2-h sample, the occurrence of which was significantly more common among aged individuals. These data indicate an age-related decline in DNA repair competence among a small subpopulation of lymphocytes.

Basal DNA damage in individual human lymphocytes with age.

A role for DNA damage is central to many theories of aging, but attempts to show an increase in DNA damage with age have yielded contradictory results. However, previous experiments have been of limited sensitivity, only able to examine induced (not basal) damage or pooled (not individual) cells. In this report, we apply a novel technique (Singh et al., 1988) to directly measure basal levels of DNA single-strand breaks and alkali-labile sites in individual human peripheral blood lymphocytes (PBL) obtained from young (less than 60 years) and old (more than 60 years) male donors. This approach shows that while average changes with age are small, changes in certain individuals and in certain cells may be large: the mean increase in damage was only 12%, but the increase in a subpopulation of highly damaged lymphocytes was 5-fold. However, most of this increase was contributed by just 3 of 17 older subjects. Further characterization of these individuals may shed light on the relationship between DNA damage and aging.

Changes in fibronectin mRNA splicing with in vitro passage.

Senescent human fibroblasts produce larger fibronectin molecules with altered binding properties. To determine if this change could involve alternative splicing of fibronectin precursor mRNA, we developed an approach using reverse transcription and the polymerase chain reaction to study fibronectin mRNA splicing at each of the three alternatively spliced regions. Two of the three regions showed changes with in vitro passage incorporation of the ED-A region increased 8 fold.

Aging--causes and defenses.

Recent studies have shown that chromosomes shorten as cells divide and become senescent. Also, deletions in mitochondrial DNA increase markedly with advancing age, presumably secondary to damage from oxygen radicals. Since host defenses against environmental factors also become attenuated, the molecular damage associated with aging may increasingly perturb normal homeostasis and increase the susceptibility to disease and disability. Such age-associated dysfunctions can be targeted and interrupted.

Construction of DNA recognition sites active in Haemophilus transformation.

Competent Haemophilus cells recognize and preferentially take up Haemophilus DNA during genetic transformation. This preferential uptake is correlated with the presence on incoming DNA of an 11-base-pair (bp) sequence, 5'-A-A-G-T-G-C-G-G-T-C-A-3'. To prove that this sequence is the recognition site that identifies Haemophilus DNA to the competent cell, we have now constructed a series of plasmids, each of which contains the 11-bp sequence. Using two different assay systems we have tested the ability of fragments from these plasmids to compete with cloned Haemophilus DNA fragments that naturally contain the 11-bp sequence. We find that the addition of the 11-bp sequence to a DNA fragment is necessary and sufficient for preferential uptake of that fragment. However, plasmid DNAs containing this sequence may vary as much as 48-fold in uptake activity, and this variation correlates with the A+T-richness of the DNA flanking the 11-mer.

Expression of prohibitin in rat seminiferous epithelium.

Subtractive hybridization was used to isolate cDNAs highly expressed in stages IX-XI of the cycle of the seminiferous epithelium in the rat. One of the cloned cDNAs was sequenced and shown to be homologous to a previously described cDNA encoding rat prohibitin. Northern blot analyses showed that 1.9- and 1.2-kb transcripts were present in Sertoli cells whereas 1.5-, 1.2-, and 0.7-kb transcripts were expressed in germ cells. Western blot analyses with anti-peptide antibody to prohibitin revealed only a single 30-kDa protein in testis. Immunocytochemistry demonstrated that prohibitin protein was expressed constitutively in adult Leydig cells and Sertoli cells at all stages. Immunoreactivity of prohibition was very low in preleptotene spermatocytes, very high in leptotene spermatocytes, and very low in zygotene spermatocytes. In pachytene spermatocytes, immunoreactivity was very high in stages VII-XI and was minimal during stages XII and XIV. No protein was detected in spermatogonia and spermatocytes undergoing mitotic and meiotic divisions, respectively. These studies show that the prohibitin gene is expressed differentially in testis. The expression pattern of the prohibitin gene in rat testis appears to correlate with a proposed antiproliferative role of prohibitin.

Increased susceptibility to SV40 transformation with development and in vitro aging.

The incidence of most cancers increases with aging. To examine whether this increased risk might be related to a higher susceptibility of older cells to neoplastic transformation, we transfected rat fibroblasts aged in vivo and in vitro with origin-defective SV40 DNA and measured the number of transformed foci. Substantial increases in the number of transformed foci were observed in cells from adult rats when compared with those of cells from embryos or weanlings. Much higher numbers of foci were also obtained at late passage, when 68% or more of the in vitro lifespan had been completed, while no foci were produced from cells at early or middle passage. To control for changes with aging in uptake, integration, or expression of exogenous DNA, parallel cultures were transfected with a G418 resistance gene. The number of G418-resistant colonies did not increase with aging and, in fact, decreased in late passage embryonic cell cultures. Therefore, increased susceptibility to SV40 transformation appears to be a feature of development and in vitro aging in rat cells.

Identification of estrogen receptor mRNA and the estrogen modulation of parathyroid hormone-stimulated cyclic AMP accumulation in opossum kidney cells.

The opossum kidney (OK) cell was used as a model to test the hypothesis that estrogen directly affects proximal renal tubular epithelial cells. To demonstrate the expression of estrogen receptor in OK cells, we developed an approach using reverse transcription and the polymerase chain reaction. Analysis of the DNA amplified with nested primers revealed the predicted size fragment and restriction enzyme digestion products. To demonstrate the functional effects of estrogen, OK cells at confluence were preincubated in serum-free medium for 7-10 days with or without 17 beta-estradiol. Bovine PTH(1-34) (bPTH(1-34)) then stimulated a dose-dependent intracellular accumulation of cAMP that was maximal after 1 min and then gradually declined. Cyclic AMP in the medium slowly increased over 60 min. Preincubation with 17 beta-estradiol did not affect cell proliferation as measured by total protein content but caused an inhibition of bPTH(1-34)-stimulated intracellular cAMP accumulation that was maximal at 10(-11) M 17 beta-estradiol (71 +/- 3% control, p less than .001). bPTH(1-34) also increased cAMP release into the medium, an effect maximal using 10(-10) M 17 beta-estradiol (118 +/- 3% control, p less than .001). Preincubation with the inactive isomer 17 alpha-estradiol caused no changes in cAMP accumulation or release. Coincubation with the antiestrogen tamoxifen blocked the effects of 17 beta-estradiol. Sodium-dependent phosphate transport was: (1) inhibited by 2-h incubations with 10(-8) or 10(-10) M bPTH(1-34) and not affected by preincubation with 17 beta-estradiol, and (2) not inhibited by a 20-min incubation with 10(-8) M bPTH(1-34) unless cells were preincubated with 10(-8) M 17 beta-estradiol, suggesting that any possible effects of estrogen on phosphate transport are not directly mediated by changes in cAMP. These studies demonstrate the presence of estrogen receptor mRNA in OK cells as well as direct and specific effects of physiologic concentrations of estrogen on cAMP accumulation in these cells. This system may be a good model for further study of estrogen and PTH effects on the kidney.

Primitive neuroectodermal tumor arising in the pancreas.

Peripheral primitive neuroectodermal tumors (PNETs) are extra cranial primitive small round blue cell tumors showing histologic, immunohistochemical or electron microscopic evidence of neuroectodermal differentiation. They are most commonly encountered in the soft tissue or bone in children and young adults. We report an unusual case of a PNET arising in the pancreas. A 17-yr-old male presented with a pancreatic mass and underwent a pancreatoduodenectomy. Histologically, the neoplasm was composed of sheets of small round cells that stained for cytokeratin, neuron specific enolase, and 12E7 but not muscle specific action, desmin, common leukocyte antigen, chromogranin, synaptophysin, or other islet cell markers. The diagnosis of PNET in this unusual location was confirmed by cytogenetic analysis which showed the chromosomal translocation characteristics of PNETs, t(11;12)(q24;q12). This case highlights the difficulty in the diagnosis of PNET when it is present in visceral organs where other neuroendocrine neoplasms and adenocarcinomas are more common.