Intrahypothalamic implantation of progesterone in castrated male whiptail lizards (Cnemidophorus inornatus) elicits courtship and copulatory behavior and affects androgen receptor- and progesterone receptor-mRNA expression in the brain.

A primary tenet of behavioral neuroendocrinology is that gonadal steroid hormones act on limbic nuclei to activate mating behavior in vertebrates. Traditionally, research has focused on the regulation of male-typical sexual behavior by testicular androgens and female-typical sexual behavior by ovarian estrogen and progesterone. Indeed, progesterone generally is regarded as an antiandrogen, acting centrally to inhibit sexual behavior in males. However, experiments with lizards, and more recently with rats, have challenged this paradigm. For example, exogenous progesterone induces mating behavior in some, but not all, castrated male whiptail lizards. The present study determined that implantation of progesterone into the anterior hypothalamus preoptic area of castrated, progesterone-sensitive males completely restored sexual behavior but failed to elicit sexual activity in castrated, progesterone-insensitive males. Further, androgen receptor -and progesterone receptor-mRNA expression in specific brain regions was significantly different in progesterone-sensitive versus progesterone-insensitive animals. Progesterone-sensitive males showed significantly higher relative abundance of androgen receptor-mRNA in the preoptic area, amygdala, and lateral septum, as compared with progesterone-insensitive animals receiving the same treatment. In contrast, progesterone receptor-mRNA abundance was lower in preoptic area of progesterone-sensitive males than in progesterone-insensitive males. No differences were found in the baseline abundance of androgen receptor-or progesterone receptor-mRNA in these nuclei between control groups of progesterone-sensitive and progesterone-insensitive males who were castrated but not implanted. This suggests that progesterone differentially regulates its own receptor as well as androgen receptor in areas of the brain involved in the control of sexual behavior of males and that the nature of this regulation shows individual variability.

Cell adhesion molecules as targets for Hox genes: neural cell adhesion molecule promoter activity is modulated by cotransfection with Hox-2.5 and -2.4.

In an effort to determine whether homeobox genes modulate the activity of the promoter of the mouse neural cell adhesion molecule (N-CAM) gene, we have carried out a series of cotransfection experiments using NIH 3T3 cells. Plasmids were constructed containing Xenopus laevis Hox-2.5 and -2.4 coding sequences linked to a human cytomegalovirus promoter (CMV-Hox-2.5 and CMV-Hox-2.4). A 4.9-kilobase DNA fragment containing 5' flanking and first exon sequences of the mouse N-CAM gene was linked to a chloramphenicol acetyltransferase (CAT) reporter gene (N-CAM-Pro-CAT). Cotransfection with CMV-Hox-2.5 and N-CAM-Pro-CAT resulted in a strong induction of CAT activity. The N-CAM promoter contained two potential homeodomain binding sites (sites I and II) within a 47-base-pair segment (512-559 base pairs upstream of the ATG codon in the first exon of the N-CAM gene). This segment was linked to a minimal promoter (simian virus 40 early) and a downstream CAT gene. Although this construct was transcriptionally active at a low level in NIH 3T3 cells, cotransfection of CMV-Hox-2.5 resulted in CAT activity that was greatly elevated. Mutational studies revealed that it was the homeodomain binding site II sequence that was required for this regulation. In contrast, cotransfection with CMV-Hox-2.4 eliminated the CAT activity that was driven by the CMV-Hox-2.5 construct. Thus, the products of two related Hox genes, which are located adjacent to each other in the Hox-2 complex, can differentially modulate transcription from the promoter of a cell adhesion molecule gene. The results suggest that the N-CAM gene is likely to be a target for regulation by Hox gene products.

Four exons encode a 93-base-pair insert in three neural cell adhesion molecule mRNAs specific for chicken heart and skeletal muscle.

The neural cell adhesion molecule (N-CAM) is detected in chicken brain as three polypeptides of 180 kDa, 140 kDa, and 120 kDa that arise from a single gene by alternative splicing. Heart tissue, however, contains components of 150 kDa, 140 kDa, and 130 kDa; neither the differences in molecular mass among these components nor the difference between neural and cardiac N-CAM could be accounted for by variations in glycosylation alone. A cDNA clone isolated from an embryonic chicken heart library, [lambda N101B, 1.8 kilobases (kb)] contained a 93-base-pair (bp) insert not found in neural N-CAM cDNAs. In the N-CAM gene this sequence mapped within a large region between exons 12 and 13 and was derived from four exons (12A-D) of 15, 33, 42, and 3 bp. Exons 12C and 12D together coded for 15 amino acids very similar to the second half of the muscle-specific insert (MSD1) found in N-CAM cDNA from human muscle cell cultures [Dickson, G., Gower, H. J., Barton, C. H., Prentice, H. M., Elsom, V. L., Moore, S. E., Cox, R. D., Quinn, C., Putt, W. & Walsh, F. S. (1987) Cell 50, 1119-1130]; the sequences of 12A and 12B, however, were much less similar to the corresponding region of the MSD1 sequence. Two oligonucleotides, one specific to exons 12A plus 12B and one specific to exon 12C both recognized mRNA species of 6.4 kb, 4.3 kb, and 3.0 kb in chicken cardiac and skeletal muscle and no mRNA species in smooth muscle or brain. The 3' end of clone lambda N101B contained a sequence coding for a potential phosphatidylinositol linkage signal as does the smallest form of brain N-CAM. In heart cell membranes only the 130-kDa N-CAM polypeptide was released by phospholipase C, suggesting that this form of N-CAM is encoded by clone lambda N101B. The other heart N-CAM species (150 kDa and 140 kDa) may be transmembrane forms that include the 12A-D (and possibly other) inserts. Tissue-specific forms of N-CAM can thus be formed by alternative use of multiple small exons that may alter the conformation of the extracellular region of the molecule. Differential use or switching of these small exons in conjunction with the differential expression of larger exons specifying regions associated with the cell membrane and cytoplasmic domains may signal key events in embryogenesis and histogenesis.

Neural cell adhesion molecule: structure, immunoglobulin-like domains, cell surface modulation, and alternative RNA splicing.

The neural cell adhesion molecule, N-CAM, appears on early embryonic cells and is important in the formation of cell collectives and their boundaries at sites of morphogenesis. Later in development it is found on various differentiated tissues and is a major CAM mediating adhesion among neurons and between neurons and muscle. To provide a molecular basis for understanding N-CAM function, the complete amino acid sequences of the three major polypeptides of N-CAM and most of the noncoding sequences of their messenger RNA's were determined from the analysis of complementary DNA clones and were verified by amino acid sequences of selected CNBr fragments and proteolytic fragments. The extracellular region of each N-CAM polypeptide includes five contiguous segments that are homologous in sequence to each other and to members of the immunoglobulin superfamily, suggesting that interactions among immunoglobulin-like domains form the basis for N-CAM homophilic binding. Although different in their membrane-associated and cytoplasmic domains, the amino acid sequences of the three polypeptides appear to be identical throughout this extracellular region (682 amino acids) where the binding site is located. Variations in N-CAM activity thus do not occur by changes in the amino acid sequence that alter the specificity of binding. Instead, regulation is achieved by cell surface modulation events that alter N-CAM affinity, prevalence, mobility, and distribution on the surface. A major mechanism for modulation is alternative RNA splicing resulting in N-CAM's with different cytoplasmic domains that differentially interact with the cell membrane. Such regulatory mechanisms may link N-CAM binding function with other primary cellular processes during the embryonic development of pattern.

Cell surface modulation of the neural cell adhesion molecule resulting from alternative mRNA splicing in a tissue-specific developmental sequence.

The neural cell adhesion molecule N-CAM is an intrinsic membrane glycoprotein that is expressed in the embryonic chicken nervous system as two different polypeptide chains encoded by alternatively spliced transcripts of a single gene. Because they differ by the presence or absence of approximately 250 amino acids in their cytoplasmic domains, these polypeptides are designated ld and sd, for large and small cytoplasmic domain, respectively. We report here that the ld-specific sequences comprise a single exon in the chicken N-CAM gene and that developmental expression of the ld and sd chains occurs in a tissue-specific fashion, with the ld chain restricted to the nervous system. Comparison of the nucleotide sequences from an N-CAM genomic clone with cDNA sequences showed that a single exon of 783 base pairs corresponded to the unique cytoplasmic domain of the ld polypeptide. Sequences from this exon were absent from the single N-CAM mRNA detected in several non-neural tissues by RNA blot hybridization, and immunoblot analysis confirmed that antigenic determinants unique to the ld-specific domain were not expressed in these tissues. Immunohistochemical experiments indicated that only the sd chain was expressed on cell surfaces of non-neural tissues throughout embryonic development. The ld chain was found on cell bodies and neurites of differentiated neurons; it first appeared as neurons began to extend neurites and to express the neuron-glia cell adhesion molecule (Ng-CAM) and it was restricted to definite layers in laminar tissues such as the retina and cerebellum. These results suggest that the control of mRNA splicing may affect the regulation of N-CAM function at specific sites within the nervous system and thus influence the control of neural morphogenesis and histogenesis.

Alternatively spliced mRNAs code for different polypeptide chains of the chicken neural cell adhesion molecule (N-CAM).

Rabbit polyclonal antibodies directed against the chicken neural cell adhesion molecule (N-CAM) were used to isolate four overlapping cDNA clones from a chicken cDNA expression library in bacteriophage gamma gt11. These clones collectively accounted for 3.8 kilobases of N-CAM mRNA sequence and hybridized specifically to two 6-7-kilobase brain polyadenylated RNA species that co-migrated with previously identified N-CAM mRNAs. DNA fragments derived from an internal region of the cloned cDNA sequences hybridized to the larger but not to the smaller N-CAM mRNA species, while fragments on either side of this region hybridized to both mRNAs. A cDNA fragment that recognized only the larger mRNA was subcloned into gamma gt11, and the expressed fusion protein was used to affinity-purify rabbit polyclonal antibodies; the antibodies recognized only the larger of the two structurally related N-CAM polypeptides. In contrast, when several cDNA clones that recognized both mRNAs were used to purify antibodies, the antibodies recognized both polypeptides. The results, in conjunction with other data indicating that there is one gene specifying N-CAM, suggest that different N-CAM polypeptides are synthesized from multiple N-CAM messages generated by alternative splicing of transcripts from a single N-CAM gene.

Isolation of a cDNA clone for the liver cell adhesion molecule (L-CAM).

Liver cell adhesion molecule (L-CAM) is a calcium-dependent cell adhesion molecule found in very early vertebrate embryos and on liver and other epithelial cells in adults. To describe the genes coding for the molecule and study its synthesis, we have cloned cDNA from poly(A)+ RNA of 10-day embryonic chicken liver using the delta gt11 expression vector. One clone, lambda L301, has been characterized and used in analyses of L-CAM mRNA and genomic DNA. Clone lambda L301 produced a fusion protein that reacted strongly with polyclonal antibodies that recognize L-CAM (Mr 124,000) and its Mr 81,000 NH2-terminal fragment, Ft1, released from liver membranes by trypsin. This result indicates that lambda L301 contains a cDNA insert complementary to protein coding sequence within the two-thirds of the mRNA coding region beginning at the 5' end. The 220-base-pair cDNA insert was isolated and used as a probe in hybridization experiments. RNA transfer blot analysis of poly(A)+ RNA showed a single 4-kilobase mRNA; Southern blot analysis showed multiple components consistent with the presence of one to three L-CAM genes. To test whether different tissues express different forms of L-CAM message, poly(A)+ RNA from eight embryonic organs was analyzed. Only organs that expressed L-CAM protein contained poly(A)+ RNA that hybridized to the lambda L301 probe; in all cases a single band, with the same mobility as that in liver, was observed. The L-CAM mRNA in each tissue was present in proportions similar to those detected previously for the L-CAM protein in these tissues. The combined results suggest that any possible heterogeneity in the L-CAM genes is not reflected in the size of either the mRNA or protein.

Minichromosomal repetitive DNA in Trypanosoma cruzi: its use in a high-sensitivity parasite detection assay.

We have isolated genomic clones containing members of a tandemly repeated DNA family from Trypanosoma cruzi. This family, which contains a 195-base pair (bp) repeating unit, is the most abundant repetitive DNA in this organism. DNA sequencing analysis of three adjacent tandem repeats as well as two independent nonadjacent repeats showed relatively little sequence heterogeneity. Surprisingly, the three tandem elements contained a 585-bp open reading frame. However, blot hybridization of RNA from epimastigotes as well as blood-form trypomastigotes failed to show evidence for transcription of these sequences. Fractionation of whole T. cruzi DNA in sucrose gradients or in agarose gels followed by hybridization with appropriate radioactive probes showed that the size distribution of DNA bearing the 195-bp repetitive element is distinct from that of kinetoplast DNA as well as from that of DNA bearing tubulin genes. Hybridization of the 195-bp element probe with DNA from six different T. cruzi strains was positive; hybridization with DNA of other protozoa was negative with the single exception of Leptomonas collosoma , which displayed a weak cross-hybridization signal. Clones bearing this repetitive element are shown to be useful as probes for identification and counting of T. cruzi cells by dot-blot hybridization. The sensitivity of this assay permits detection of the DNA of 30 parasites in blood samples.