Regulation of human cardiac myosin heavy chain genes: the effect of catecholamine.

The 5'-flanking regions of the alpha- and beta-cardiac myosin heavy chain (MyHC) genes were excised from the cosmid human genomic clones using Hind III and Xbal for the alpha-MyHC gene, and the Hind III and Hind III sites for the beta-MyHC gene. These fragments were linked to chloramphenicol acetyl transferase (CAT) vector to generate a chimeric fusion gene. These fusion genes were subsequently transfected to neonatal rat cardiac cultured cells to analyze the CAT activity. The alpha-MyHC gene is preferentially expressed as compared to the beta-MyHC. In the presence of norepinephrine (NE) the beta-MyHC gene is remarkably induced (within 24 hours following the addition of norepinephrine to the cardiocyte culture). However, the alpha-MyHC is also induced. Specific alpha andrenergic antagonists such as terazosin (Tz) partially suppressed both the alpha- and beta-MyHC genes as revealed by the CAT activity. These findings suggest that catecholamine does activate the human cardiac MyHC genes but does not differentiate the specific expression of either the alpha- or beta-MyHC genes.     

Ig VH, DH, and JH germ-line gene segments linked by overlapping cosmid clones of rabbit DNA

Overlapping cosmid clones of rabbit germ-line DNA containing VH, DH and JH gene segments were isolated. The map of this cluster of cosmid clones indicated that the rabbit VH and JH regions were separated by 63 kb. Hybridization of Southern blots of these cosmid clones with two different DH segment probes identified a total of six DH segments within the region between the VH and JH regions. The nucleotide sequences of the JH region and one of the DH segments have been determined. The DH segment has conserved heptamer and nonamer sequences separated by 12 and 11 bp at the 3' and 5' sides, respectively, of the coding region and hence, appears to be a functional gene. The nucleotide sequence of the JH region revealed four functional JH gene segments and one JH pseudogene. Inasmuch as the JH region had previously been linked by contiguous overlapping clones with C mu, C gamma, C epsilon, and one C alpha gene, this VH-DH-JH cluster and the clones containing the Ig H chain C region genes represent 190 kb of contiguous germ-line DNA of the Ig H chain locus.     

Dispersed localization of D segments in the human immunoglobulin heavy-chain locus.

We have studied the organization of the human immunoglobulin heavy-chain genes by pulse field gel electrophoresis as well as by isolation of cosmid clones. The total length of the heavy-chain variable region locus was estimated to be approximately 3000 kb. We found that D segments including a recently isolated D5 segment were dispersed among VH segments. We identified a pseudo V segment 18 kb 3' to the D5 segment in isolated cosmid clones. A 300 kb fragment produced by MluI digestion contained VH, D, JH segments and the distance between VH and D was estimated to be approximately 240 kb. Overlapping cosmid clones containing the human D1, D2, D3, D4, JH, Cmu and C delta genes were isolated. Restriction maps of these regions indicated that the distance between D and JH is about 22 kb. A partial restriction map of the VH locus was constructed using the pulse field gel electrophoresis technique and deletion of VH segments in B cells.     

Molecular and functional characterization of genes encoding horse MHC class I antigens

Sequence and functional analyses were undertaken on two cDNAs and a genomic clone encoding horse major histocompatibility complex (MHC) class I molecules. All of the clones were isolated from a single horse that is homozygous for all known horse MHC class I and class II antigens. The two cDNAs (clones 8-9 and 1-29) were isolated from a lymphocyte library and encode polymorphic MHC antigens from two loci. The genomic cosmid clone, isolated from a sperm library, contains the 8-9 gene. All three genes were expressed in mouse L-cells and were recognized by alloantisera and, for the cDNAs, by alloreactive cytotoxic T lymphocytes. A total of 3815 bp of the genomic clone were sequenced, extending from 429 bp upstream (5') of the leader peptide through the 3' untranslated region. Promoter region motifs and an intron-exon structure characteristic of MHC class I genes of other species were found. A subclone containing 407 bp of the promoter region was inserted into a chloramphenicol acetyl transferase reporter plasmid, tested in transient transfection assays, and found to have promoter activity in heterologous cells. This genomic clone will enable detailed studies of MHC class I gene regulation in horse trophoblasts, and in horse retroviral infections.     

Studies on the structures of the normal and abnormal goat thyroglobulin genes

We have cloned the thyroglobulin (Tg) gene of normal goats and goitrous goats which have a Tg synthesis defect. At the 5'-end of the gene, we studied cosmid clones covering a region from 20 kilobases (kb) upstream from the Tg gene to 42 kb into it. Electron microscopy and restriction mapping show that this part of the gene contains 20 exons of 90-1190 bp, in total 4.9 kb of exonic information (56% of the mRNA) split by 19 introns of 150-9100 bp. The exons comprise 12% of the 5' sequences cloned. At the 3'-end, 55 kb were cloned, containing 10 kb of the gene which comprises only 3 exons of 550 bp in total. Sequence analysis of the 3'-end of the normal and abnormal Tg genes has revealed one transition mutation 3' to the reading frame in a stem-loop structure region of the last exon near the poly(A) addition site. Analysis of the promoter site and the first 5 exons has revealed only one difference between the normal and goitrous Tg genes: a Ser----Leu transition in exon 5. We also found an insertion in the fifth intron of the abnormal gene.     

SB subregion of the human major histocompatibility complex: gene organization, allelic polymorphism and expression in transformed cells

The SB region of the human major histocompatibility complex (MHC) has been cloned from cosmid and lambda phage libraries made from the human B-lymphoblastoid cell line Priess (DR4/4, DC4/4, SB3/4). Two alpha genes and two beta genes are encoded in the 100 kb long SB region in the order SB alpha-SB beta-SX alpha-SX beta. The SB alpha and SB beta genes encode the alpha and beta subunits of the SB subset of class II MHC molecules. Both the SX alpha and the SX beta genes are pseudogenes in the haplotype examined. From the isolated clones, the two haplotypes of the Priess cell line, SB3 and SB4, are distinguished by nucleotide sequencing and blot hybridization analyses. Restriction site polymorphisms between the SB3 and SB4 clones were observed only in relatively small regions of the SB beta and SX beta genes. A mouse macrophage cell line was transfected with one of the cosmid clones containing both SB alpha and SB beta genes. Expression of the alpha and beta genes was detected by fluorescene-activated cell sorting (FACS) and two-dimensional gel electrophoresis using SB-specific monoclonal antibodies.     

Isolation and characterization of beta- and gamma-crystallin genes from rat genomic cosmid libraries

Two libraries, together containing about 10(6) colonies, have been constructed by cloning different size fractions of a partial Sau3A digest of rat genomic DNA in the cosmid vector pTM. Upon screening with two cDNA clones, one containing alpha A2-crystallin and one containing beta B1-crystallin sequences, 14 cosmid clones were isolated which were beta B1-crystallin-specific; none was found which contained alpha A2-crystallin sequences. The inserts of the beta B1 clones, which range from 35 to 45 kb in length, contain overlapping DNA segments covering more than 60 kb of rat genomic DNA. The composite BamHI restriction map of this region shows a single beta B1-crystallin gene, which is interrupted by several intronic sequences. Five recombinants hybridizing with two different rat lens gamma-crystallin cDNA clones were also isolated from these libraries. Four of these contain 31- to 41-kb inserts, whereas the fifth recombinant contains a 12.2-kb insert. Hybridization analysis with 5' and 3'-specific cDNA fragments indicates that altogether these inserts contain six gamma-crystallin genes, three of which are located on one insert of only 31 kb.     

Comparative anatomy of the primate major histocompatibility complex DR subregion: evidence for combinations of DRB genes conserved across species.

The class II region of the human major histocompatibility complex (HLA) is made up of three major subregions designated DR, DQ, and DP. With the aim of gaining an insight into the evolution and stability of DR haplotypes, a total of 63 cosmid clones were isolated from the DR subregion (Gogo-DR) of a western lowland gorilla. All but one of these cosmid clones were found to fall into two clusters. The larger cluster, A, was defined by 41 overlapping cosmid clones and contained a DRB gene segment made up of exons 4 through 6 and four DRB genes, designated Gogo-DRB6, Gogo-DRB5*01, Gogo-DRB8, and Gogo-DRB3*01. The total length of this cluster was approximately 180 kb. The second cluster, B, encompassed a contiguous DNA stretch of approximately 145 kb and was composed of 21 overlapping cosmid clones. Cluster B contained three DRB genes, designated Gogo-DRB1*08, Gogo-DRB2, and Gogo-DRB3*02. One cosmid clone (WP1-9) containing a DRB pseudogene could not be linked to either cluster A or B. Neither the organization of cluster A nor that of cluster B was identical to that of known HLA-DR haplotypes. However, two gorilla DRB genes, Gogo-DRB6 and Gogo-DRB5*01, the human counterparts of which are linked in the HLA-DR2 haplotype, were found to be located next to each other in cluster A. The arrangement of the Gogo-DRB genes in cluster B, which is presumed to be the gorilla DR8 haplotype, was similar to that of HLA-DR3/DR5/DR6 haplotypes and to that of the presumed ancestral HLA-DR8 haplotype.(ABSTRACT TRUNCATED AT 250 WORDS)