Structure and expression of a cluster of human hematopoietic serine protease genes found on chromosome 14q11.2

We previously identified a cluster of hematopoietic serine protease genes on chromosome 14 at band q11.2. This cluster contains the cathepsin G gene and the two related cathepsin G-like genes CGL-1 and CGL-2. The CGL-1 gene is identical with the cytotoxic T cell serine protease CSP-B (also called SECT, and in mice, CCP1, granzyme B, or CTLA-1). In this report, we determined that CGL-2 is identical with a recently described gene called h-CCPX. The coding sequences of CG, CGL-1, and CGL-2 are 65-75% identical at the DNA level. The intervening sequences are much less conserved, except for introns 3 of the CGL-1 and CGL-2 genes, which are 93% identical. Each of the genes has the same overall organization, with 5 exons and 4 introns, very short 5' untranslated regions, and identical splice phases for all of the introns. Cathepsin G is expressed at high levels in promyelocytes/promonocytes, and CGL-1/CSP-B is expressed at high levels in activated cytolytic T cells, lymphokine-activated killer (LAK), and natural killer (NK) cells. CGL-2/h-CCPX is expressed at much lower levels in activated peripheral blood lymphocytes, LAK and NK cells. To begin to define the regulatory elements that target expression of each of these genes to their specific lineages at specific times, the 5' flanking region of each gene was sequenced. The 5' flanking regions are minimally related and have few conserved consensus elements. Further experiments will be required to determine the critical cis-acting regulatory sequences required for tissue- and development-specific expression of each of these genes.     

Comparison of class I (H-2) gene sequences. Derivation of unique probes for members of this multigene family

The genes for the classical transplantation antigens are unique in that they belong to a multigene family of which each member is represented by a large number of alleles. Since all of these genes are highly related in sequence, it has been difficult to study the expression of individual members of this complex gene family. Based upon our initial suggestion that the 3' noncoding regions of these genes may be useful in identifying mRNA molecules transcribed from different loci, we have compared a large number of sequences from different inbred mouse strains and have been able to assign each of these sequences without ambiguity into distinct allelic series. Such accurate assignment has afforded the opportunity to compare the coding regions of these highly homologous genes and has led to the identification of sequences which are apparently unique to specific genes in the family. Synthetic oligonucleotides corresponding to each of the locus-specific unique regions have been used successfully to type a panel of cDNA sequences, as well as to quantitate the relative amounts of mRNA transcribed from distinct loci. The availability of these specific coding probes will allow the analysis of individual genes and their specific expression without interference from other highly homologous sequences in this multigene family.     

Evolution of the casein multigene family: conserved sequences in the 5'' flanking and exon regions.

The rat alpha- and bovine alpha s1-casein genes have been isolated and their 5' sequences determined. The rat alpha-, beta-, gamma- and bovine alpha s1-casein genes contain similar 5' exon arrangements in which the 5' noncoding, signal peptide and casein kinase phosphorylation sequences are each encoded by separate exons. These findings support the hypothesis that during evolution, the family of casein genes arose by a process involving exon recruitment followed by intragenic and intergenic duplication of a primordial gene. Several highly conserved regions in the first 200 base pairs of the 5' flanking DNA have been identified. Additional sequence homology extending up to 550 base pairs upstream of the CAP site has been found between the rat alpha- and bovine alpha s1-casein sequences. Unexpectedly, the 5' flanking promoter regions are conserved to a greater extent than both the entire mature coding and intron regions of these genes. These conserved 5' flanking sequences may contain potential cis regulatory elements which are responsible for the coordinate expression of the functionally-related casein genes during mammary gland development.     

Histone genes inPhysarum polycephalum: Transcription and analysis of the flanking regions of the two H4 genes

The histone H4 multigene family of Physarum polycephalum consists of two genes, H41 and H42. Both genes have an unusual structure in that they are interrupted by a small intron. The structure of the P. polycephalum H4 genes is discussed and compared to the structure of histone genes of other organisms. S1 nuclease analysis was used to map the 5' and 3' ends of the histone H4 messengers. We show that the histone H4 genes have a hybrid structure; they are interrupted by an intervening sequence, as in replacement variant histone genes of higher eukaryotes, but their 5' and 3' noncoding regions have the properties of replication-dependent histone genes: the 5' and 3' leader and trailer sequences are short, possess a 3'-hyphenated dyad symmetry element, and a CAGA sequence is found 3' to the hyphenated hairpin structure. This report also provides evidence that both genes are expressed in late G2 phase as well as in S phase and that their expression is temporally coordinated and quantitatively similar during the cell cycle.     

Gene families encoding phase- and size-variable surface lipoproteins of Mycoplasma hyorhinis

A prototype family of seven genes encoding the variable surface lipoproteins (Vlps) of Mycoplasma hyorhinis is characterized in the pathogenic SK76 strain, using long-range PCR to amplify and analyze the single chromosomal region containing expressed genes vlpA to -G, each of which is subject to phase and size variation. Smaller families of vlp genes in subclones of SK76 or in another strain of M. hyorhinis, GDL, can be attributed to deletions of specific vlp genes from the prototype array described here. Two genes, vlpA and the newly revealed vlpG, contain repeat motifs in their 3' coding regions that differ from the short tandem repeats in other vlp genes yet retain structural features common to all vlp gene products. SK76 and GDL vlp gene families are similarly organized and show sequence similarity between corresponding individual vlp genes. In light of the extensive potential for diversity within the vlp gene system, such conservation provides a provisional basis to hypothesize that vlp genes may exist in specific arrays that endow selected functions while retaining common structural features required during phase-variable expression of this set of gene products.     

Expression of human alpha-tubulin genes: interspecies conservation of 3'' untranslated regions.

To examine the sequence complexity and differential expression of human alpha-tubulin genes, we constructed cDNA libraries from two unrelated tissue types (epidermis and fetal brain). The complete sequence of a positively hybridizing alpha-tubulin clone from each library is described. Each is shown to represent an abundantly expressed gene from fetal brain and keratinocytes, respectively. Although the coding regions are extensively homologous (97%), the 3' untranslated regions are totally dissimilar. This property has been used to dissect the human alpha-tubulin multigene family into members bearing sequence relatedness in this region. Surprisingly, each of these noncoding regions shares very high (65 to 80%) interspecies homology with the 3' untranslated region of one of the two rat alpha-tubulin genes of known sequence. These unexpected homologies imply the existence of selective pressure on the 3' untranslated regions of some cytoskeletal genes which maintains sequence fidelity during the course of evolution, perhaps as a consequence of an as yet unidentified functional requirement.     

DNase I-defined chromatin configuration of the human CD3 gene cluster.

The three CD3 genes on human chromosome 11q23 encode proteins (gamma, delta and epsilon) which form part of the antigen receptor on T lymphocytes. All three genes are clustered within 50 kb and are activated approximately contemporaneously during the early stages of T cell ontogeny. In order to pinpoint potential regulatory sequences important for locus activation and tissue-specific gene expression, the chromatin structure of almost 90 kb of this region has been probed in five cell lines using the endonuclease pancreatic DNase I. A set of DNase I hypersensitive (HS) sites has been defined in T cell chromatin, five of which were strong and not found in non-T cells, with the exception of the erythroleukaemia cell line K562, in which three sites were weakly expressed, correlating with a low level of delta mRNA. The subset of five HS sites map close to the CD3 genes and lie in regions which contain elements of defined function: the gamma promoter; the delta promoter and its 3' enhancer; and the epsilon promoter and its 3' enhancer. Since no further major T cell-restricted HS sites lie within the 90kb of the CD3 locus analysed, these five regions may contain all the sequences important for CD3 gene expression.